CN1761484A - A method of diagnosis and treatment - Google Patents

Abstract

The present invention relates generally to a method for detecting an aberrant cell, and more particularly an apoptotic cell, in a subject or in a biological sample from said subject, and agents useful for same. The presence of the aberrant cell or group of aberrant cells provides an indication of a particular disease or condition or a propensity for development of a disease or condition. More particularly, the present invention contemplates a method for detecting an apoptotic cell by detecting the presence of extranuclear nuclear molecules, in particular La, or a relative increase in extranuclear nuclear molecule levels. The present invention further provides a method for diagnosing or monitoring conditions characterised by aberrant, unwanted or otherwise inappropriate cellular apoptosis in a subject or in a biological sample from said subject by screening for up-regulation of extranuclear nuclear molecule levels in a cell or group of cells. The present invention provides diagnostic agents useful for detecting these molecules. Such diagnostic agents include immunointeractive molecules, such as antibodies.

Description

The method of diagnosis and treatment

Invention field

The present invention relates generally to the experimenter or from described experimenter's biology sample detection abnormal cell, more especially the programmed cell death cell method and be used for the reagent of this purpose.The existence of abnormal cell or abnormal cell group provides disease or the specified disease of disease or the indication of disease or tendency has taken place.More specifically, the present invention relates to by detecting the existence of outer karyon molecule, particularly La of karyon, perhaps the relative increase of the outer karyon molecular level of karyon detects the method for programmed cell death cell.The present invention further provides by the up regulation of the outer karyon molecular level of karyon in screening cell or the groups of cells and diagnose or monitor the experimenter or from the method that is the symptom of feature with unusual, that do not expect or other unsuitable cell programmed cell death in described experimenter's the biological sample.The invention provides the diagnostic reagent that is used to detect these molecules.Such diagnostic reagent comprises immunological molecule, for example antibody.

The invention still further relates to method external or the interior therapeutic orientation.The present invention further provides with experimenter's molecule on the epitope specificity interactional antibody, particularly monoclonal antibody that exist.The ability of directed programmed cell death cell is useful, particularly in the scope that the diagnosis that exists for feature with the programmed cell death cell, immunization therapy and immunoprophylaxis are handled.

Background of invention

The author describes alphabet sequence in detail at the publication bibliography of this description middle finger, and to be embodied in description last.

In this description not and should not be regarded as the knowledge or any type of prompting of Australian common practise prior art form part the reference of prior art.

Encroach on normal structure with malignant tumor or cancer that uncontrolled mode is grown, and shift and growth away from the place of former tissue through being everlasting.Generally speaking, cancer is called not knowing of its process fully or having only several normal cells to produce of vicious transformation from experience.Cancer almost can produce from any tissue in the health.From epithelial cell produce be called cancer those are modal cancer kinds.Sarcoma is the malignant tumor from the stroma that resembles the such cell generation of fibroblast, muscle cell and adipose cell.The solid malignant of lymphoid tissue is called lymthoma, and the malignant tumor of the bone marrow of lymphocyte and other hematopoietic cells and generation blood is called leukemia.

In industrialized country, cancer is dead three roughly therefore one.Because the treatment of infectious disease and the control of cardiovascular disease improve constantly, and prolong life expectancy, cancer may become these national modal fatal diseases.Therefore, need successfully treat cancer, remove or destroy all malignant cells, and do not make death.An immunne response that desirable approach is an inducing antitumor that achieves this end is treated tumor cell and their corresponding normal cells with a certain discrimination.Yet the trial of immunization method that surpasses the treatment cancer in a century all is unsustainable.

Therefore, treat method for cancer at present and continue to use surgical excision (if possible), if desired, then carry out the method for radiotherapy and/or this life-time service of chemotherapy.The success rate mobility of this very jejune form of therapy is very big, and because tumor further develops and shifts success rate is reduced greatly usually.In addition, these treatments are relevant with serious adverse, comprise the cicatrix (for example mammectomy or amputation) that disfigurement and operation form, serious nausea and vomiting, chemotherapy, and the most tangible, damage to normal structure, for example to hair follicle, internal organs and bone marrow, this is that non-specific relatively directional mechanism result as the toxic medicament of most of treatments of cancer parts causes.

In addition, most of anticancer therapies comprise that cytotoxicity chemotherapeutics, signal conduction depressant drug, radiotherapy, monoclonal antibody and cytotoxic lymphocyte are by the programmed cell death kill cancer.Though tumor can comprise a certain proportion of programmed cell death cell, even the necrosis of certain area taking place before the anticancer disease treatment, respond anticancer disease treatment, occurs the programmed cell death cell number and the more large-area necrosis that increase in the tumor in advance.Yet, when cytotoxicity chemotherapy material is used for the treatment of terminal cancer, often be difficult to estimate degree and the response of tumor that cell kills to treating for the first time.As usual, the patient has accepted three cycle chemotherapy at least before the tumor response is estimated in clinical and radiation.Usually, minority patient with advanced cancer pair cell drug toxicity responds, and the patient can experience the treatment side effect that does not obtain benefit like this.Therefore, for can be fast after in advance in respect of the period 1 treatment of therapeutic response, diagnostic method convenient and that detect tumor-killing (it often then estimates survival rate) reliably has does not have satisfied needs of medical treatment.For example, the adenocarcinoma of esophagus patient who accepts chemotherapy radiotherapy before performing the operation is used fluoro-deoxyglucose (FDG-PET) positron emission X tomography x, with＞90% susceptiveness and specificity difference treatment respondent and respondent not, be easy to predict that those people experience effective excision of its tumor subsequently.Learn tumor whether in early days response can make Most patients need not carry out invalid and have a toxic treatment.Then, the patient carries out second round treatment or the clinical trial of the medicine studied to not responding.

Therefore, for the new method of developing with oriented approach diagnosis and treatment cancer urgent and continuous demand is arranged.Up to now, effective orientation is killed and wounded the also not realization of idea of malignant cell.

In implementing work of the present invention, record astoundingly, use interacting molecule, immunological molecule for example, in fact can reliably and can screen the karyon molecule with detecting, and the method that accurately and reliably detects the programmed cell death cell in external and the body in the high degree of specificity mode further is provided.Especially, diagnosis and the frequent feature that exists for certain proportion programmed cell death cell of monitoring to tumor and transfer are advanced now.Further, determine now to use interacting molecule of the present invention to help carrying out antineoplaston at high orientation and specificity.

Summary of the invention

An aspect of of the present present invention relates to the experimenter or from the method for described experimenter's biology sample detection programmed cell death cell, described method comprises that the interacting molecule of using at karyon molecule or its antigen part contacts described experimenter or forms from the cell of described experimenter's biological sample or cell extract and screening interacting molecule-karyon molecular complex, and the non-karyon location of wherein said complex is the indication of programmed cell death cell.

Another aspect of the present invention relates to the experimenter or from the method for described experimenter's biology sample detection programmed cell death cell, described method comprises that the immunological molecule of using at karyon molecule or its antigen part contacts described experimenter or forms from the cell of described experimenter's biological sample or cell extract and screening immunological molecule-karyon molecular complex, and the non-karyon location of wherein said complex is the indication of programmed cell death cell.

Another aspect of the present invention provides to the experimenter or from the method for described experimenter's biology sample detection programmed cell death tumor cell, described method comprises that the immunological molecule of using at La or its antigen part contacts described experimenter or forms from the cell of described experimenter's biological sample or cell extract and screening immunological molecule-La complex, and the non-karyon location of wherein said complex is the indication of programmed cell death tumor cell.

Another aspect of the present invention provides to the experimenter or from the method for described experimenter's biology sample detection programmed cell death tumor cell, described method comprises that the antibody of using at La or its antigen part contacts described experimenter or forms from the cell of described experimenter's biological sample or cell extract and screening antibody-La complex, and the non-karyon location of wherein said complex is the indication of programmed cell death tumor cell.

Another aspect of the present invention relates to experimenter's diagnosis or monitors with unusual method that do not expect or that other unsuitable cell programmed cell death is the symptom of feature, described method comprises the karyon molecule used at described karyon molecule or its antigenic determinant or epi-position-contact described experimenter or from the cell or the cell extract of described experimenter's biological sample in conjunction with the interacting molecule of effective dose, and qualitatively or quantitatively determine karyon molecule-immunological molecule complex and form, the non-karyon location of wherein said complex is the indication of cell programmed cell death.

Another aspect of the present invention relates to the method to experimenter's diagnosis or monitoring tumor disease, described method comprises using at the La-of described La or its antigenic determinant or epi-position and contacts described experimenter in conjunction with the immunological molecule of effective dose or from the cell or the cell extract of described experimenter's biological sample, and qualitatively or quantitatively determining La-immunological molecule complex forms, the non-karyon location of wherein said complex is the indication of cell programmed cell death, and the described cell programmed cell death indication that is described tumor disease.

The invention further relates to the analytical method of programmed cell death cell in the detection of biological sample, described method comprises following step :-

(1) interacting molecule at karyon molecule or its antigenic determinant is contacted with suspecting the biological sample that contains described karyon molecule; With

(2) complex that forms in the step (1) is carried out signal detection step,

Wherein detecting non-karyon interacting molecule-karyon molecular complex formation is the indication of programmed cell death cell.

Another aspect of the present invention relates to the method to people detection programmed cell death cell, described method comprises to described patient introduces the interacting molecule at karyon molecule or its antigenic determinant of using the reporter molecule labelling, make the interacting molecule of labelling introduce the interacting molecule propagation of selecting part at blood circulation, then described patient is carried out the reporter molecule detection means and detect to determine the interacting molecule position by blood circulation or to described patient.

Another aspect of the present invention provides the method to sample detection La or its fragment, variant or derivant, comprise the formation that makes sample contact antibody or its fragment or derivant and detection comprise the complex of described antibody and La or its fragment, variant or derivant, wherein the non-karyon of La location is the indication of programmed cell death.

The invention further relates to interacting molecule at the karyon molecule be used for preparing detection from patient's biological sample programmed cell death cell quantitatively or the purposes of sxemiquantitative diagnostic kit, described test kit can have operation instruction and can be form automatic or automanual or that be complementary with automation or software.

It is the method for the disease of feature with the cell programmed cell death that another aspect of the present invention relates to experimenter's therapeutic and/or prophylactic treatment, described method is included in to be enough to treat under the time and condition of described disease, described experimenter is used the interacting molecule at karyon molecule or its antigen part of effective dose, and described interacting molecule is connected with the effector structure, bonding or association.

The more special method that provides experimenter's therapeutic and/or prophylactic treatment tumor disease of the present invention, described method be included in be enough to suppress, reduce or in addition decrement regulate under the time and condition of tumor growth, described experimenter is used the immunological molecule at La or its antigen part of effective dose, and described immunological molecule is connected with the effector structure, bonding or association.

Method to patient treatment treatment metastatic carcinoma is provided on the other hand, described method be included in be enough to suppress, reduce or in addition decrement regulate under the time and condition of described metastatic carcinoma growth, described experimenter is used the immunological molecule at La or its antigen part of effective dose, and described immunological molecule is connected with the effector structure, bonding or association.

Another aspect of the present invention relate to the effector structure link coupled anti--karyon interaction of molecules molecule is used to prepare the purposes of the medicine of treatment patient disease, described disease is a feature with the cell programmed cell death, and wherein said effector structure is treated described disease.

On the other hand, the present invention relates to contain the pharmaceutical composition of regulator defined above and one or more pharmaceutical acceptable carriers and/or diluent.Described regulator is called active component.

Another aspect of the present invention relates to the medicament defined above when using in the method for the invention.

The accompanying drawing summary

Fig. 1 is with or without the graphic representation that serum is cultivated 24 hours prostate cancer cell line LNCaP before being used for flow cytometry analysis in that cell is scraped from tissue culture flasks.Cell dyes with iodate third ingot (PI), and (A) annexin V-FITC, (B) the people La autoantibody (hLa) and the mouse anti-human IgG-FITC of normal human serum (NHS) or affinity purification, (C) Mus isotype tester or mouse-anti-hLa mAb clone SW3 and anti--mice IgG-FITC, (D) Mus isotype tester or mouse-anti-hLa mAb clone 3B9 and anti--mice IgG-FITC.Rectangular histogram shows the cell (be late programmed cell death cell) of sorting (gated) as the PI intermediate.Blue line, NHS or Mus isotype tester; The black line is to annexin V or anti--La of the LNCaP cell dyeing of growing in the serum; Red line is to annexin V or anti--La of the LNCaP cell dyeing that do not have serum growth.

Fig. 2 is the graphic representation of anti--La function.In case the programmed cell death (Fig. 2 A) of anti--inductive cancerous cell of La antibody diagnosis chemotherapy, they can then send other modalities of cancer treatment, its with dead cell near those effective non-cross tolerance mechanism of cancerous cell (Fig. 2 B) of surviving of keeping.Shown in Fig. 2 A is for the first time after the chemotherapy, and anti--La antibody (yellow) detects programmed cell death cancerous cell (lead), and it is near survival growth of cancer cells (light gray).Shown in Fig. 2 B is anti--La antibody (purple), and it has non-cross tolerance anticancer therapy, sends for the killing and wounding of the cancerous cell of keep living (bystander killing) (redness).

Fig. 3 describes that the programmed cell death body is external to be formed and gradually in conjunction with the graphic representation of anti--La antibody.Using 0.5 μ M D-82041 DEISENHOFEN (STS) is inducing apoptosis to the Jurkat human T cell leukemia cell.3B9 and the dyeing of karyon impermeability (impermeant) nucleic acid combination dye iodate third ingot (PI) pair cell with the FITC labelling.Arrow represents that the programmed cell death body is along with the time forms gradually.The quadrant vernier is set to isotype tester Sal5＜3% dyeing.

Fig. 4 describes with programmed cell death Jurkat cell is bonded to resist-graphic representation that La antibody is relevant with cumulative membrane permeability.Use 0.5 μ M STS to Jurkat cell induction programmed cell death.Anti--'beta '-tubulin mAb (tubulin) and the dyeing of PI pair cell with the Sal5 (isotype tester) of FITC-labelling or 3B9 of FITC-labelling (anti--La antibody) or FITC-labelling.

Fig. 5 be with programmed cell death Jurkat cell bonded anti--graphic representation of the time course of La antibody.Use 0.5 μ M STS to Jurkat cell induction programmed cell death.Anti--'beta '-tubulin mAb (tubulin) trypan blue pair cell dyeing with the Sal5 (isotype tester) of FITC-labelling or 3B9 of FITC-labelling (anti--La antibody) or FITC-labelling.Shown in the longitudinal axis is the total cell percentage ratio of each time point that is positive to each indicator dyeing.Data use Minimum Variance method to make curve (B) with single line mapping (A) or with Prism v3.0.

Fig. 6 be describe the programmed cell death body and with the programmed cell death body bonded anti--La antibody is external to be stable graphic representation.Use 0.5 μ M STS to Jurkat cell induction programmed cell death.3B9 and iodate third ingot (PI) (row of going up) pair cell dyeing with the FITC-labelling.Use forward direction scatterplot (FSC) and side scatterplot (SSC) (row down) to confirm the size and the inner complexity of programmed cell death body respectively.

Fig. 7 describes annexin V, and 7AAD and anti--La antibody and programmed cell death cell are combined in the external programmed cell death process with time correlation and along with the graphic representation of time change.Use 0.5 μ M STS to Jurkat cell induction programmed cell death.With people's annexin V (annV-FITC) of FITC-labelling, 3B9 of R-phycoerythrin-labelling (PE-3B9) and the dyeing of 7AAD pair cell.

Fig. 8 describes annexin V during the external programmed cell death, and the time dependence combination of 7AAD and anti--La antibody and programmed cell death cell is relevant graphic representation.Use 0.5 μ M STS to Jurkat cell induction programmed cell death.With people's annexin V (annV-FITC) of FITC-labelling, 3B9 of R-phycoerythrin-labelling (3B9) and the dyeing of 7AAD pair cell.Incident in the R1-3 district (event) (one group on left hand) is analyzed size (forward direction scatterplot or FSC) and inner complexity (side scatterplot or SSC) (middle a group) and with 7AAD and 3B9 dye (one group on the right hand).

Fig. 9 comprises describing and resists-La antibody and bonded image of downright bad Jurkat cell and graphic representation.By heating 1 hour down to the necrosis of Jurkat cell induction at 56 ℃.A. use 3B9 (anti--La antibody) (green) and the karyon impermeability DNA-combination dye of Alexa488-labelling, the dyeing of 7-amino-actinomycin D (7AAD) (redness) pair cell, or B. dyes with the 3B9 (anti--La antibody) (green) of Alexa488-labelling and annexin V (redness) pair cell of R-phycoerythrin-labelling, and by laser scanning with the video picture of focus microscope.C. use the Sal5 (isotype tester) of FITC-labelling or the 3B9 of FITC-labelling (anti--La antibody) and PI pair cell to dye and analyze by flow cytometer.

Figure 10 describes anti--La antibody preferential and late programmed cell death cell and the bonded graphic representation of programmed cell death body.Use 0.5 μ M STS to Jurkat cell induction programmed cell death.3B9 and the dyeing of PI pair cell with the FITC-labelling.Behind the apoptosis inducing 20 hours or 48 hours, to 3B9 with PI difference dyeing (1 in the left hand column and 2) ⁺Subgroup is divided and being selected to the some feature analysis that looses.Scatter plot (right hand analysis) shows the 3B9 of accumulation in time ⁺PI ^IntermediateAct on less (according to the low size of forward direction scatterplot [FSC] mensuration) and granule less (according to the inside complexity that reduces of forward direction scatterplot [SSC] mensuration) (following right hand group).The quadrant vernier is arranged on isotype tester Sal5 dyeing＜3%.

Figure 11 is that to describe anti--La antibodies be that caspase (caspase) 3 is dependent and form relevant graphic representation with the programmed cell death body.With expressing enhanced independent GFP egfp (EGFP) or the plasmid DNA carrier transient transfection MCF-7 cell of former caspase 3 and EGFP.Use the MCF-7 cell induction programmed cell death of 0.5 μ M STS, and dye with the 3B9 and the 7AAD pair cell of FITC-labelling to transient transfection.The cell that shows in the scatterplot (left hand figure) sorting process on green fluorescence.Then, the incident in the quadrant 1 and 2 (left hand figure) is used for the scattering signatures analysis by sorting process in each scatter diagram.Scattering analysis (right hand group) shows that the 3B9 combination is that caspase 3 is dependent, therefore with the programmed cell death body form relevant because programmed cell death body less (the low size of measuring according to forward direction scatterplot [FSC]) and than granule (the inside complexity of the reduction of measuring according to forward direction scatterplot [SSC]) (following right hand group).The quadrant vernier is arranged on isotype tester Sal5 dyeing＜3%.

Figure 12 comprises that describing anti--La antibodies is that caspase 3 is dependent and form relevant imaging and graphic representation with the programmed cell death body.With vehicle Control thing (B) or express the carrier stable transfection MCF-7 cell of former caspase 3 (C and C ').Handle 24 hours to MCF-7 transfectant execution programmed cell death with 1 μ MSTS.A. for flow cytometry analysis, with the 3B9 and the dyeing of iodate third ingot (PI) pair cell of Alexa488-labelling.B, C and C '.For the fluorescence microscope analysis, with the 3B9 (green) and the dyeing of karyon dyestuff DAPI (blueness) pair cell of Alexa488-labelling.Indicate programmed cell death body (arrow).

Figure 13 is the image of anti--La antibody dead cell kytoplasm carrying capacity.Handle 24 hours to Jurkat cell induction programmed cell death with 0.5 μ M STS.With karyon impermanency dyestuff TOPRO3 (blueness), the 3B9 of Alexa488-labelling or anti--La antibody, the dyeing of people's annexin V (redness) pair cell and the use of isotype tester Sal5 or anti--PARP mAb (green) and R-phycoerythrin (PE)-labelling are observed with the focal argon laser scanning microscope.Following A ' is observed in the 3B9 dyeing of A.Alexa488-labelling. than high power; Observe negative isotype contrast for the anti--La antibody staining that uses Sal5; C. anti--PARP dyeing.

Figure 14 describes anti-other karyons and antigenic other monoclonal antibodies of ribose karyon also in conjunction with the image of programmed cell death body.Handle 24 hours to Jurkat cell induction programmed cell death with 0.5 μ M STS.With anti--α-fodrin mAb (green) of Alexa488-labelling and 7AAD (redness) pair cell dyes and by laser scanning with the focus microscopic examination.

Figure 15 describes to compare with programmed cell death initial stage T cell, and after the pernicious Jurkat T cell programmed cell death, La/SS-B expresses the graphic representation of up regulation.The peripheral blood lymphocytes of ficoll-purification (PBMC) was cultivated 4 days in the RPMI-1640 that 10% hyclone is arranged, and handled with 1 μ M STS at last 24 hours that cultivate then.Similarly, last 24 hours that cultivate with 1 μ M STS inducing apoptosis before with T cell mitogen conconavalin A (PBMC-ConA) 10 mcg/ml m with PBMC activation 4 days.Handle 24 hours to Jurkat cell (Jurkat) inducing apoptosis with 0.5 μ M STS.The quadrant vernier is arranged on isotype tester Sal5 dyeing＜3%.

Figure 16 describes anti-other karyons and antigenic other monoclonal antibodies of ribose karyon also in conjunction with the graphic representation of programmed cell death cell.With 0.5 μ M STS to Jurkat cell induction programmed cell death.MAb pair cell dyeing with PI and various FITC-labellings: isotype tester, Sal5, for anti--La/SS-B clone 3B9 (anti--La antibody), anti--'beta '-tubulin clone TUB2.1 FITC conjugate (Sigma F 2043), proliferative cell karyon antigen (PCNA) clone PC10 (Oncogene Cat #NA03), mouse anti-α-fodrin (non-redness resists-spectrin) Chemicon MAB1622, anti--lamen B clone 101-B7 (oncogene cat#NA12) and anti--PARP clone C2-10 (oncogene cat #AM30).The quadrant vernier is arranged on corresponding isotype tester antibody staining＜3%.

Figure 17 describes anti-other karyons and antigenic other monoclonal antibodies of ribose karyon also in conjunction with the graphic representation of programmed cell death body.With single expression EGFP or-the plasmid DNA carrier transient transfection EGFP MCF-7 cell of former caspase 3 and EGFP.With the MCF-7 cell induction programmed cell death of 0.5 μ M STS to transient transfection, and isotype mAb with 7AAD and FITC-labelling, Sal5 and anti-La/SS-B mAb (3B9), lamin B and the dyeing of proliferative cell karyon antigen (PCNA) pair cell.Above one group of MCF-7 cell (branch is elected the EGFP-positive as) of representing not have transfection.Do not have transfection the MCF-7 cell outward appearance with the MCF-7 cell (not providing data) of the dna vector transfection of single expression EGFP much at one.

Figure 18 be describe by the people anti--the La autoantibody detects the graphic representation of programmed cell death people cell.By make with 0.5 μ M STS the cell of Jurkat cell generation programmed cell death use through the people of La-affinity purification anti--the Mus mAb 3B9 of La autoantibody (above a group) or anti-people La (below a group) dyeing.PI ⁺Cell sorting and data are represented with the rectangular histogram of each time point after the apoptosis inducing.Negative control, human IgG (thick line); The people resists-hLa antibody and 3B9 (fine rule).

Figure 19 describes the graphic representation that another kind of Anti-Human La/SS-B monoclonal antibody also detects programmed cell death people cell.For the Jurkat cell, perhaps in In vitro culture, do not handle (being untreated) or handle 17 hours inducing apoptosis (processing) with 0.5 μ M STS.Cell resists or mAb clone SW3 or mAb clone 3B9 dyeing with the anti--Mus two of PI and FITC-labelling.

Figure 20 describes anti--La antibody and the bonded graphic representation of initial stage programmed cell death cell from the Rodents species.Do not replenish (A, D), perhaps add 1 μ M dexamethasone (B, E) or 0.5 μ M STS (C, F) under, external Mus (A-C) or rat (D-F) thymocyte cell were cultivated 21-24 hour.Cell dyes with the isotype mAb of FITC-labelling, 3B9 of Sal5 (contrast) or FITC-labelling (anti--La antibody) and iodate third ingot (PI).

Figure 21 describes anti--La antibody and the bonded graphic representation of programmed cell death tumor cell from the Rodents species.Mus thymus lymphoblast cell line, EL-4 (A-E) and rat prostate cancerous cell line, AT-3.1 (F) and 0.5 μ M STS (A, F), etoposide (B) or etoposide and cyclophosphamide (C), In vitro culture (A-C, F) 24 hours, perhaps in (D) that never handles or the body with cyclophosphamide and etoposide handle 48 hours the induced tumor programmed cell death (E) the hypodermic implant of homology C57BL/6 mice reclaim the EL-4 tumor cell.Cell dyes with the isotype mAb of FITC-labelling, 3B9 of Sal5 (contrast) or FITC-labelling (anti--La antibody) and iodate third ingot (PI).

Figure 22 describes anti--La antibody and multiple programmed cell death people and bonded graphic representation of monkey tumor cell line.Cell line was handled inducing apoptosis: A.Jurkat T chronic myeloid leukemia 24 hours with 0.5-1 μ M STS; The B.U2OS osteosarcoma cell; The C.HeLa cervical cancer cell; The D.MG63 osteosarcoma cell; E.COS-7 monkey kidney fibroblast.Cell dyes with the isotype mAb of FITC-labelling, 3B9 of Sal (contrast) or FITC-labelling (anti--La antibody) and iodate third ingot (PI).

Figure 23 describes by the diagram of the programmed cell death process in various external stages.After programmed cell death stimulates, programmed cell death cellular contraction and fragmentation film forming bound fraction, known is the programmed cell death body, it leaks gradually along with the time or is downright bad once more.At last, the programmed cell death body resolves into oligoneucleosomes, and then is decomposed into free DNA.

Figure 24 passes through as usual with annexin V and for example diagram description in the programmed cell death stage of 7AAD dyeing definition of the permanent dyestuff of karyon.Handled 16 hours with 0.5 μ M D-82041 DEISENHOFEN,, and dye with annexin V (AV) and 7-amino-actinomycin D (7AAD) to Jurkat cell induction programmed cell death.1, living cells (AV ^-, 7AAD ^-); 2, early stage programmed cell death cell (AV ⁺, 7AAD ^-); 3, late period programmed cell death cell (AV ⁺, 7AAD ⁺).

Detailed description of the present invention

The present invention is that part proposes according to following wonderful discovery: the use immunological molecule screens the outer karyon developed by molecule of karyon, and high degree of specificity and the reliable method of the existence that detects the programmed cell death cell in external or the body is provided. This finds particular importance, because all do not identify karyon molecule, particularly La towards the experiment work (thereby identifying antigen) of the screening antigen relevant with the programmed cell death cell in the past, is candidate antigens. Really, other incoherent antigens, for example the phosphatidyl serine has been identified repeatedly. Regrettably, these incoherent molecules show remarkable defective, and particularly owing to non-programmed cell death event, for example cell is mechanical or other are broken, and instantaneous extracellular expression can occur. Also have, in the past to think that cell dyeing is that the result of the active process of internalization of the cell surface complex of some programmed cell death cell antagonist-antigens has been excluded the mark as programmed cell death itself as karyon molecule, for example La of fundamental analysis. Namely, got rid of membrane permeability enters cell as antibody a approach. Therefore, also do not develop the method for identifying programmed cell death cell itself. Yet, develop now to use and detect programmed cell death cell and the method for programmed cell death body in late period in the external and body of anti--karyon immunological molecule, particularly anti--La/SS-B immunological molecule. Described method relates to but does not require additional step, and such as the osmosis of proof combination, this needed before the present invention occurs. Known resisting-karyon antibody was in conjunction with it doesn't matter with apoptosis inducing in the past, it depends on the osmosis of cell, in conjunction be skin covering of the surface relevant and depend on that bonding mechanism rather than the passive antigentic specificity that enters and comprise be combined in the programmed cell death body.

The karyon molecule for example La be accredited as astoundingly the programmed cell death cell high specific, can detect and exclusive mark, allow now exploitation to relate to a series of reagent and the method for diagnosis and monitoring programmed cell death cell mass, particularly tumour and their transfer. In addition, determine to use immunological molecule for these karyon molecules to provide for targeted therapy and/or preventative-therapeutic high specific method with the illness that exists for feature of programmed cell death cell. Particularly, the importance of the oncotherapy certain content programmed cell death cell that is to find to make anti--La immunological molecule to be oriented in the tumour for example can successfully be used to provide the method that for example realizes looking on (bystander) non-programmed cell death tumor cytotoxicity by sending toxic molecule.

Therefore, one aspect of the present invention relates to the experimenter or from the method for described experimenter's biology sample detection programmed cell death cell, described method comprises uses cell or cell extract and the screening interacting molecule-karyon molecular complex formation that contacts described experimenter or described biological sample for the interacting molecule of karyon molecule or its antigen part, and the non-karyon location of wherein said complex is the indication of programmed cell death cell.

More specifically, the present invention relates to the experimenter or from the method for described experimenter's biology sample detection programmed cell death cell, described method comprises uses cell or cell extract and the screening immunological molecule-karyon molecular complex formation that contacts described experimenter or described biological sample for the immunological molecule of karyon molecule or its antigen part, and the non-karyon location of wherein said complex is the indication of programmed cell death cell.

" the karyon molecule " that relates to should be appreciated that and refer to any protein or the nonprotein molecule in the permanent or instantaneous karyon that is present in the cell that becomes the programmed cell death main body before the programmed cell death. Preferably, described karyon molecule is Ro52, Ro60, La/SS-B, gelsolin, α-fodrin, fibrillarin, U1 micronuclear ribonucleoprotein matter (U1 snRNP), heteronuclear ribonucleoprotein matter (hnRNP), lamin B, poly-(ADP-ribose) polymerase (PARP), PCNA (PCNA), SC-35 splicing factor, Smith (Sm) antigen. More preferably, described karyon molecule is La.

Not in office where face limits the present invention, and is anti--La antibody specific detection programmed cell death. The non-karyon location of the previous La that understands and subsequently detect on programmed cell death outside surface according to the programmed cell death specificity as a result caspase (caspase) activate and propose. Yet, although can't detect caspase activity (P.Smolewski etc., J Immunol Methods 2002) in the programmed cell death cell late, determine the karyon molecule for example La still can be detected. Be not easy to detect programmed cell death cell in late period by other diagnostic methods.

Should be appreciated that about " non-karyon location " and to refer to be positioned at any district of cell or its part rather than the experimenter's karyon molecule within intact cell nuclear. Preferably, the non-karyon of experimenter location so, so that La is exposed to born of the same parents' external environment, here be called " born of the same parents' outside fix ", during generation, for example, the karyon molecule translocates in the kytoplasm of programmed cell death cell there, described cell membrane becomes permeability or express there this molecule in the programmed cell death body, and wherein the programmed cell death body forms in the programmed cell death cell and is distributed in born of the same parents' external environment when the programmed cell death cell decomposes fully at last.

Should be appreciated that the cell that refers to experience or experienced programmed cell death about " programmed cell death " cell. The present invention is not limited by any theory or the mode of action, and programmed cell death is the activation that needs dying cell metabolic activity. Frequently, programmed cell death is characterised in that the contraction of cell, and dna cleavage becomes fragment (providing " ladder pattern " at gel) and is chromatinic condensation and limit. The cell programmed cell death occurs in a lot of situations. Therefore, the evaluation of the non-karyon location of La, for example provide together monitoring with the character of the cell of expressing this molecule and position and/or diagnosed the method for a variety of illnesss, comprise miocardial infarction (heart attack) or brain (apoplexy), perhaps autoimmunity and other inflammation diseases, perhaps virus disease, AIDS for example, or neurodegenerative disease, for example alzheimer's disease or Parkinson's, perhaps acute solid organ or bone-marrow transplantation rejection, the perhaps tissue damage (' catarrh ') or the tumour that cause of chemotherapy or radiotherapy. In a preferred embodiment, experimenter's programmed cell death cell is the programmed cell death tumour cell.

According to this preferred embodiment, provide to the experimenter or from the method for described experimenter's biology sample detection programmed cell death tumour cell, described method comprises cell or cell extract and the screening immunological molecule-La complex formation that contacts described experimenter or described biological sample with the immunological molecule that points to La or its antigen part, and the non-karyon location of wherein said complex is the indication of programmed cell death tumour cell.

Preferably, described non-karyon location occurs in the kytoplasm of programmed cell death cell or within the plastidogenetic programmed cell death body of programmed cell death.

Should be appreciated that about " oncocyte " and to refer to show unusual dead cell. Term " growth " should and comprise with its broad understanding and refer to breed. With this viewpoint, the example of abnormal cell growth is the uncontrolled propagation of cell. Oncocyte can be benign cell or malignant cell. Experimenter's oncocyte can be any cell type, for example epithelial cell or non-epithelial cell.

The common medical meaning of term " neoplasia " refers to " new Growth of Cells ", and it is as to normal growth control, for example to the result of the forfeiture of growth of tumour cell responding ability.

" propagation " refers to experience the cell of the unusual high speed of growth. Yet according to used herein, term " neoplasia " and " hyperplasia " can Alternates, refer generally to experience the cell of abnormal cell growth speed. Neoplasia and hyperplasia comprise " lump ", and it can be optimum, before pernicious or pernicious. Term " knurl " should be appreciated that be on the health damage, tumour or other encirclements or do not have material block or other growth forms of surrounding, comprise tumour cell.

According to used herein, term " super propagation " and " neoplasia " Alternate and refer to abnormality or take fast breeding or neoformation as the illness of feature in those cells. The term meaning is cell, tissue or the organ that comprises all types of cancer growths or oncogene pathology, transfer tissue or vicious transformation, and does not consider Histology or intrusion state. " the super propagation of pathologic " cell exists in the morbid state take malignant growth as feature.

Those skilled in the art's recognized term " cancer " and refer to epithelium or the malignant tumour of endocrine tissue comprises respiratory system carcinoma, gastrointestinal system cancer, genitourinary system carcinoma, carcinoma of testis, breast cancer, prostate cancer, internal system cancer and melanoma. The cancer that exemplifies comprises those that form from breast tissue. This term also comprises carcinosarcoma, for example comprises the malignant tumour of cancer and sarcoma organizational composition. " gland cancer " refer to cancer that glandular tissue produces or wherein tumour cell generate discernible glandular structure.

Term used herein " knurl " comprises all terms of discussing in three sections of the fronts.

The example of knurl of the present invention and neoplasia cell includes but not limited to the central nervous system knurl, retinoblastoma, neuroblastoma and other paediatrics knurls, head and neck cancer (for example squamous cell carcinoma), breast and prostate cancer, lung cancer (ED-SCLC and non-small cell lung cancer), kidney (for example renal cell adenocarcinoma), oesophagus cancer of the stomach, hepatocellular carcinoma, the ductus pancreaticus biloma forms (for example gland cancer pancreatic endocrine knurl), colorectal cancer, cervical carcinoma and cancer of anus, uterus and other genital tract cancers, carcinoma of urethra (for example ureter and carcinoma of urinary bladder), blastocyte tumour (for example testis blastocyte tumour or ovary blastocyte tumour), oophoroma (for example epithelial ovarian cancer), unknown primary carcinoma, human immune deficiency associated malignancies (for example Kaposi ' s sarcoma), lymthoma, leukaemia, chromoma, sarcoma, endocrine knurl (for example thyroid adenoma), celiothelioma and other pleurals tumor, neuroendocrine tumor and cancer sample tumour.

Here comprise the form of ownership that refers to La about " La ", perhaps its homologue or homologue or derivative. Should be appreciated that it is to comprise referring to replace any isotype that montage produces from the La mRNA of La or variant or multiform variant about " La ". Should also be appreciated that " La " is the molecule of the term SS-B that substitutes.

" interacting molecule " is any molecule that La or its antigen part or its homologue or derivative is had specificity (must not be exclusive specificity, but exclusive specificity being preferred) and binding affinity. The example of interacting molecule comprises immunity bioactive molecule and peptide mimics matter. Although preferred immunological molecule is a kind of immunoglobulin molecules, the present invention extends to other immunological molecules, antibody fragment for example, and single-chain antibody goes immune antiboidy to comprise humanized antibody and T-cell related antigen-binding molecule (TABMs). Most preferably, immunological molecule is a kind of antibody, for example polyclonal antibody or monoclonal antibody. Should be appreciated that the main body immunological molecule can with any other protein or nonprotein molecule or cell be connected, bonding or associate in addition. Most preferably, antibody is monoclonal antibody.

Interacting molecule be " at " the karyon molecule, for example La perhaps, is the degree of immunological molecule to interacting molecule.Should be appreciated that molecule must not be exclusive specific, but exclusive specificity is preferred.For example, known antibodies sometimes with other antigenic cross-reactions.Antigenic determinant or epi-position comprise the part that can produce immunoreactive molecule.Antigenic determinant or epi-position can be the positions of B-cell epitope or suitable T-cell receptors binding molecule.Term " antigen part " comprises antigenic determinant or epi-position.

Preferably, the main body immunological molecule is an antibody.

More preferably, described antibody is monoclonal antibody.

According to this embodiment preferred, provide to the experimenter or from the method for described experimenter's biology sample detection programmed cell death tumor cell, described method comprises uses cell or cell extract and the screening antibody-La complex formation that contacts described experimenter or described biological sample at the antibody of La or its antigen part, and the non-karyon location of wherein said complex is the indication of programmed cell death cell.

Preferably, described non-karyon is located in the kytoplasm that occurs in the programmed cell death cell or in the plastidogenetic programmed cell death body of programmed cell death.

Should be appreciated that any biological material specimens that is meant from individuality about " biological sample ", such as but not limited to, mucus, feces, urine, blood, serum, cell extract, biopsy specimen and the liquid that imports individual health and remove subsequently, for example lung lavage is afterwards from the saline solution of lung extraction or the solution that reclaims from the clysmata washing liquid.The biological sample of the method according to this invention analysis can be directly analyzed or may needs the processing of some form before being analyzed.For example, biopsy samples needed homogenization or section before molecule.

According to the present invention, suggestion programmed cell death cell, comprise that the pernicious or nonmalignant tumor cell born of the same parents of programmed cell death express La outward.Therefore, the outer qualitative or quantitative horizontal detection of La of born of the same parents cell is provided is programmed cell death with the relevant indication of disease that with the cell programmed cell death is feature.

Therefore the invention provides a kind of diagnosis or monitoring with unusual method that do not expect or that other unsuitable cell programmed cell death is the disease of feature.So-called " unusual that do not expect or unsuitable in addition " meaning is that experimenter's programmed cell death may be an excessive levels, inappropriate level or normal level, but this level is unsuitable or does not expect.According to the detailed description here, having a lot is the disease of feature there to be cell programmed cell death to a certain degree, for example, and cardiac muscle or cerebral tissue infraction, perhaps autoimmune and other inflammation diseases, perhaps virus disease, for example AIDS, or neurodegenerative disease, for example presenile dementia or parkinson disease, the rejection of perhaps acute solid organ or bone marrow transplantation, the perhaps tissue injury's (' mucositis ') or the tumor that cause of chemotherapy or radiotherapy is as tumor.

Though the preferred embodiments of the invention relate to the existence of screening programmed cell death, this is the indication that the specified disease disease starts, and also may take place wherein to its clinical scenarios of screening the reduction of cell programmed cell death level or not having programmed cell death.A kind of situation in back is for example indicating the disease of receiving treatment to exist when alleviating the state development to individuality monitoring medical treatment therapeutic scheme process and programmed cell death level.It is the indication that the disease disease takes place that the cell programmed cell death incident in some cases of should also be appreciated that does not exist.For example, in normal thymus cell development process, in positive and negative selection process, experience in the thymus of programmed cell death and have most of thymocyte cell, for take place self/non-self discriminative power this be essential.Therefore, not having the cell programmed cell death incident of normal level in the teenager thymus is the indication that child tends to take place autoimmune disorder.Therefore should be appreciated that, though the present invention may use in for the existence of the screening specific cells programmed cell death incident of the disease that can diagnose the illness in large quantities, yet the inventive method can be applied to screening lacks the programmed cell death incident, and the generation or the tendency of some disease disease takes place in this situation indication.Method of the present invention can also be used for screening the variation in the level of monitoring of diseases disease or therapeutic or prophylactic treatment scheme cell programmed cell death.

Therefore, another aspect of the present invention relates to experimenter's diagnosis or monitors with unusual method that do not expect or that other unsuitable cell programmed cell death is the disease of feature, described method comprises the karyon molecule used at described karyon molecule or its antigenic determinant or epi-position-contact described experimenter or from the cell or the cell extract of described experimenter's biological sample in conjunction with the interacting molecule of effective dose, and qualitatively or quantitatively determine karyon molecule-immunological molecule complex and form, the non-karyon location of wherein said complex is the indication of cell programmed cell death.

Preferably, described karyon molecule is La.

Preferably, described interacting molecule is an immunological molecule, more preferably is anti--La antibody, for example anti--La monoclonal antibody.

Most preferably, described non-karyon location is born of the same parents' outside fixs, and more preferably, in the kytoplasm of programmed cell death cell or in the plastidogenetic programmed cell death body of programmed cell death.

In another preferred embodiment, described disease is cardiac muscle or cerebral tissue infraction, perhaps autoimmune and other inflammation diseases, virus disease, for example AIDS, or neurodegenerative disease, for example presenile dementia or parkinson disease, acute solid organ or bone marrow transplantation rejection, tissue injury that chemotherapy or radiotherapy cause (' mucositis ') or tumor are as tumor.

In the most preferred embodiment, the present invention relates to method to experimenter's diagnosis or monitoring tumor symptom, described method comprises with the La-that points to described La or its antigenic determinant or epi-position and contacts described experimenter in conjunction with the immunological molecule of effective dose or from the cell or the cell extract of described experimenter's biological sample, and qualitatively or quantitatively determining La-immunological molecule complex forms, the non-karyon location of wherein said complex is the indication of cell programmed cell death, and the described cell programmed cell death indication that is described tumor disease.

Preferably, described tumor is central nervous system's tumor, retinoblastoma, neuroblastoma and other department of pediatrics tumors, head and neck cancer (for example squamous cell carcinoma), breast and carcinoma of prostate, pulmonary carcinoma (small cell lung cancer and nonsmall-cell lung cancer), renal carcinoma (for example renal cell adenocarcinoma), esophagus gastric cancer, hepatocarcinoma, the ductus pancreaticus biloma forms (for example adenocarcinoma pancreatic endocrine tumor), colorectal carcinoma, cervical cancer and anus cancer, uterus and other reproductive tract cancers, carcinoma of urethra (for example ureter and bladder cancer), blastocyte tumor (for example testis blastocyte tumor or ovary blastocyte tumor), ovarian cancer (for example epithelial ovarian cancer), unknown primary carcinoma, human immune deficiency associated malignancies (for example Kaposi ' s sarcoma), lymphoma, leukemia, malignant melanoma, sarcoma, endocrine tumor (for example thyroid tumor), mesothelioma and other pleural neoplasms, neuroendocrine tumor and cancer sample tumor.

Most preferably, described immunological molecule is an antibody, is more preferably monoclonal antibody.

Here should be appreciated that to comprise the people primates, livestock animals (sheep for example, pig, cattle about " experimenter ", horse, donkey), laboratory test animal (mice for example, rabbit, rat, Cavia porcellus), the wild animal of companion animals (for example Canis familiaris L., cat) and stable breeding (for example fox, kangaroo, deer).Preferably, mammal is the people.

Use antibody particularly monoclonal antibody, for example mentioned above those detect karyon molecules for example La be preferable methods of the present invention.Antibody can prepare by any method.For example, for detecting people La, people-human monoclonal antibodies can be from the B cell, the patient of anti-from producing-La autoantibody obtains this material, because they suffer from system's autoimmune disease, for example whole body lupus erythematosus (SLE) or Sjogren ' s syndrome (Lupus 1 (3): 157-165 such as Ravirajan, 1992).Antibody generally derives from non-human mammal, but not necessarily, primate for example, livestock animals (for example sheep, pig, cattle, goat, horse), laboratory test animal (for example mice, rat, rabbit, Cavia porcellus), and companion animals (for example Canis familiaris L., cat).Generally speaking, external pair cell or biopsy tissue are carried out the mensuration based on antibody.Yet,, under people's application, humanization situation, the patient is used, and measures the site that the karyon label is built up by radiologic technique with for example karyon labelling antagonist labelling if antibody is suitably gone immunity.Therefore think that La antibody is the directed reagent of cell programmed cell death.Therefore, the present invention extends to the immune form of going of the antibody that is used for people and the imaging of non-human patients cell programmed cell death.Below this is further described.

Therefore, the invention provides the antibody, particularly monoclonal antibody that is used in the body the immunoassay of La or the imaging of pair cell programmed cell death.Present obtainable antibody comprises SW3 and 3B9.

About antibody La production of antibodies, need extract this molecule from biological sample, comprise that the people organizes or from cell culture no matter be from animal by recombinant methods.Can separate La from biological sample by any suitable method.For example, separation can obtain following one or multinomial benefit: La surface charge property, size, density, biologic activity and to another individual affinity (for example with it in conjunction with or other associating another kind of protein or chemical compound).So, for example, can realize separating La by following one or more technology: super centrifugal from biofluid, ion exchange chromatography (anion-exchange chromatography for example, cation-exchange chromatography), electrophoresis (for example polyacrylamide gel electrophoresis, isoelectrofocusing), size separation (for example, gel filtration, ultrafiltration) with the affinity mediation separate that (for example immune affine separation includes but not limited to, magnetic bead separates, for example Dynabead ^TMSeparate immunochromatography, immunoprecipitation).According to the La of research or obtain the biological activity or the physical property of the tissue of La, can select the isolation technics of using.

Preferably, isolating La has kept comformational epitope on the protein from biofluid, therefore, suitably avoids causing the technology of enzyme denaturation.Those skilled in the art recognize that to keep or the importance (for example obtaining the biofluid of La) of the peculiar physiological condition of near-earth simulation La as far as possible identical with the antigenic determinant on the La that guarantees to be exposed to animal or avtive spot structure and native protein.This guarantees to produce the suitable antibody of identification native protein in the immune animal body.In preferred embodiments, utilize affine separation, one or more in gel filtration and the ultrafiltration separate La from biofluid.

Utilize standard method can carry out the generation of immunity inoculation and the monoclonal antibody followed, for example Kohler and Milstein, Nature 256:495-499,1975; Kohler and Milstein, Eur.J.Immunol.6 (7): 511-519,1976; Coligan etc., Current Protocols in Immunology, John Wiley ﹠amp; Sons, Inc., 1991-1997, or Toyama etc., " Monoclonal Antibody, ExperimentManual ", Kodansha Scientific, 1987 publication are described.In essence, use the biofluid that contains La to animal immune by standard method, preparation produces the cell of antibody, the somatic cell (for example bone-marrow-derived lymphocyte) that particularly produces antibody.Take out these from the animal of immunity then and be used for unlimited breeding.

Use the fragment of La to produce under the situation of antibody, need at first itself and carrier coupling.So-called " carrier " meaning is that the natural or artificially of the bad immunogenic substance of non-immunogenic or immunogenicity (for example hapten) is attached thereto to strengthen its immunogenic general high-molecular weight any material.

Utilize method well known in the art can produce the unlimited breeding of the cell of antibody.For example, can realize unlimited breeding (Kozbor etc., Methods in Enzymology 121:140,1986) by the method for using Epstein-Barr virus (EBV).In preferred embodiments, utilize cell fusion method that the cell that produces antibody is infinitely bred.(be described in Coligan etc., 1991-1997, above).This technology is widely used in MONOCLONAL ANTIBODIES SPECIFIC FOR.In the method, effectively producing the cell of production of antibodies body antibody (somatic antibody), particularly B cell and myeloma cell line merges.These somatic cells can come to have by oneself the people of circulation La-reactive antibody and lymph node, spleen and the peripheral blood of the preferred rodent of raw animal, for example mice and rat.Mouse boosting cell is useful especially.Yet it also is possible using rat, rabbit, sheep or goat cell or replacing from the cell of other animal species.

From lymphocytoma prepare the fusion method that specific myeloma cell line is used to prepare hybridoma (Kohler and Milstein, 1976, above; Shulman etc., Nature 276:269-270,1978; Volket etc., J.Virol.42 (1J:220-227,1982).Cultivate these cell lines at least three reasons are arranged.The firstth, myeloma cell that help never merging and unlimited similarly self-reproduction selects the hybridoma that merges.Usually, this realizes that by the myeloma that use has enzyme defect described enzyme defect makes them not grow in some selective medium of supporting the hybridoma growth.Second reason is to be produced by the capability that the lymphocyte tumor cell produces their autoantibodys.Produce tumor cell antibody in order to reduce hybridoma, use can not produce the myeloma cell line of endogenous light or heavy immunoglobulin chain.The 3rd reason selecting these cell lines is that they are for the adaptability and the efficient that merge.

A lot of hybridoma cell lines can be used for the generation of fused cell heterozygote, comprise P3X63-Ag8 for example, P3X63-AG8.653, P3/NS1-Ag4-1 (NS-1), Sp2/0-Ag14 and S194/5.XXO.Bu.1.K hler and Milstein (1976, above) P3X63-Ag8 and NS-1 cell line have been described.Shulman etc. (1978, above) developed the Sp2/0-Ag14 myeloma cell line.Trowbridge, J.Exp.Med.148 (1) .313-323,1978 have reported S194/5.XXO.Bu.1 cell line.

The preparation method that produces the spleen of antibody or lymph-node cell and myeloma cell is usually directed under the existence of the material that promotes cell membrane to merge (chemical compound, virus or) ratio difference mixture cell and the myeloma cell (but this ratio also can change from about 20: 1 to about 1: 1) with 10: 1.Fusion method is existing to be described (Kohler and Milstein, 1975, above; 1976, above; Gefter etc., Somatic Cell Genet.3.231-236,1977; Volk etc., 1982, above).The reagent that the promotion that those research worker are used is merged is Sendai virus and Polyethylene Glycol (PEG).

Because fusion method produces the heterozygote that survives (for example when spleen is used as the somatic cell source, from rough per 1 * 10 with low-down frequency ⁵Splenocyte only obtains a heterozygote), preferably have the cell that merges from not have of keeping, particularly there is not the myeloma cell of fusion to select the method for fused cell heterozygote.The method of hybridoma that detects the generation antibody of expectation from the fused cell heterozygote of other generations also is essential.Generally speaking, by supporting the hybridoma growth still to stop cultured cell in the culture medium that continues the unlimited splitted myeloma cell's of fusion growth under the normal condition, realize the selection of fused cell heterozygote.The somatic cell of using in the fusion is not retained in the medium-term and long-term viability of In vitro culture thing, does not therefore throw into question.In an embodiment of the present invention, use the myeloma cell's (HPRT-feminine gender) who does not have hypoxanthine phosphoribosyltransferase.In hypoxanthine/aminopterin-induced syndrome/thymidine (HAT) culture medium, carry out selection in the culture medium that a kind of wherein fused cell heterozygote is survived owing to the HPRT-positive gene type of splenocyte at these cells.The myeloma with different genes defective (medicine susceptiveness etc.) that use can be selected at the culture medium of supporting the effective heterozygote growth of genotype also is possible.

Selectivity is cultivated the fused cell heterozygote needs several weeks.Early stage in this period, need to identify those heterozygotes that produce the antibody of expecting, making can be then with them clone and breeding.Generally speaking, about 10% heterozygote of acquisition produces the antibody of expectation, and about 1 to about 30% scope is uncommon.By any detection that can realize the heterozygote of generation antibody in several standard method of analyses, comprise enzyme linked immunological method of testing and radiating immuning analysis technology, for example, be described in (writing) Monoclonal Antibodies andHybridomas:A New Dimension in Biological Analyses such as Kennet, pp.376-384, Plenum Press, New York, 1980, and pass through facs analysis.

In case selected the fused cell heterozygote of expectation and be cloned in each cell line that produces antibody, can breed each cell line with any of two kinds of standard methods.The suspension of hybridoma can be injected to the tissue compatible animal.The animal of accepting injection produces tumor then, the specific monoclonal antibody that its secretion fused cell heterozygote produces.Extract the body fluid of animal out, for example serum or ascites fluid provide the high concentration monoclonal antibody.Perhaps, can the test culture vessel in each cell line of external breeding.Can gather in the crops the culture that contains high concentration single specificity monoclonal antibody by decant, filtration or purification centrifugal and subsequently.

Analyze the specificity that they detect La by any suitable immunologic detection method pair cell system.For example, cell line assigned in several holes by equal portions and insulation and by enzyme linked immunological method of testing (ELISA), indirect fluorescent antibody technique, or the like, analyze the supernatant in each hole.But the evaluation generation can be discerned the cell line of the monoclonal antibody of the non-target thing of target thing La nonrecognition epi-position, and directly the histocompatibility animal is given in In vitro culture or injection then, formation tumor and generation, the antibody that collection and purification need.

These antibody are that La is specific.This means that antibody capable is distinguished La from other molecules.Can use more broad-spectrum antibody, condition is the not cross reaction of molecule in they and the normal cell.

In preferred embodiments, main body antibody is Anti-Human La monoclonal antibody, 8G3 and 9A5 (Bachmann etc., Proc Natl Acad Sci USA 83 (20): 7770-7774,1986), Anti-Human La monoclonal antibody (mAb), and LalB5 (Mamula etc., J Immunol 143 (9): 2923-2928,1989), Anti-Human La monoclonal antibody (Carmo-Fonseca etc., ExpCell Res 185 (1): 73-85,1989), Anti-Human and anti--Niu La monoclonal antibody, SW1, SW3 and SW5 (Pruijn etc., Eur J Biochem 232 (2): 611-619,1995), Anti-Human and anti--rodent La mAb, and La4B6 (Troster etc., J Autoimmunity 8 (6): 825-842,1995) or Anti-Human and anti--Mus La mAb, 3B9 (Tran etc., ArthritisRheum 46 (1): 202-208,2002) or its derivant, homologue, analog, the chemical equivalence thing, mutant or analogies.

Be also to be understood that the present invention extends to immunological molecule and expresses the cell line of main body immunological molecule, the hybridoma of particularly expressing monoclonal antibody.

When monoclonal antibody estimates to be used for in-vivo imaging or treatment, need go immunity to the host (for example people) who has imported.Go immunization method can adopt any in the various ways, comprise the preparation that has same or similar specific chimeric antibody with monoclonal antibody prepared in accordance with the present invention.Chimeric antibody is typically to be subordinated to the immune globulin variable region of different plant species and the antibody of gene constructed its light chain of constant region and heavy chain gene by genetic engineering.Therefore, according to the present invention, in case obtain to produce the hybridoma of the monoclonal antibody of expecting, then utilize technology to produce (interspecific) monoclonal antibody between kind, the land of one of them species combines (Liu etc. with the non-binding district of the antibody of another kind of species, Proc Nalt Acad Sci USA84:3439-3443,1987).For example, can be grafted on people's antibody from the complementary determining region (CDRs) of inhuman (for example Mus) monoclonal antibody, thereby with murine antibody humanization (the open No.0239400 of European patent; Jones etc., Nature 321:522-525,1986; Verhoeyen etc., Science239:1534-1536,1988; Richmann etc., Nature332:323-327,1988).In this case, removing immunization method is specific for the people.More particularly, CDRs can be grafted on the people's antibody variable region that is with or without human constant region.Provide the non-human antibody of CDRs generally to be called " donor ", provide people's antibody of framework generally to be called " receptor ".Do not need to exist constant region, if but existing, they must be substantially the same with human normal immunoglobulin's constant region, i.e. at least about 85-90%, preferably approximately 95% or more homogeneity.Therefore, except possible CDRs, all parts of humanized antibody are substantially the same with natural human immunoglobulin sequences appropriate section.Therefore, " humanized antibody " is the antibody that comprises humanization light chain and humanization heavy chain.Think donor antibody to pass through " humanization effect " process and quilt " humanization ", because the humanized antibody that expection obtains combines with antigen, it is the same with the donor antibody that CDRs is provided.Here indication " humanization " comprises that finger removes the antibody of immunity to specific host, in this case, is the human host.

Should be appreciated that immune antibody can have other conserved amino acid metalepsis, this replacement is to antigen combination or the not influence basically of other immunoglobulin functions.Can carry out conservative for example according to table 1 replaces.

Table 1

Original residue	Replacement for example
		Ala	Ser
Arg	Lys
		Asn	Gln，His
Asp	Glu
		Cys	Ser
Gln	Asn
		Glu	Asp
Gly	Pro
		His	Asn，Gln
Ile	Leu，Val
		Leu	Ile，Val
Lys	Arg，Gln，Glu
		Met	Leu，Ile
Phe	Met，Leu，Tyr
		Ser	Thr
Thr	Ser
		Trp	Tyr
Tyr	Trp，Phe
		Val	Ile，Leu

Can be used for preparing the method for giving an example of immune antibody of going according to the present invention and be described in, for example, list of references Richmann etc., 1988, above; The open No.0 239 400 of European patent; Chou etc. (U.S. Patent No. 6,056,957); Queen etc. (U.S. Patent No. 6,180,370); Morgan etc. (U.S. Patent No. 6,180,377).

Therefore, in one embodiment, the present invention relates to have for the specific immune antibody molecule that goes being arranged by the epi-position of anti-La monoclonal antibody identification, a CDRs of the wherein said variable region of going immune antibody is from the described monoclonal antibody of anti-La, and the immunoglobulin derivative moiety that goes the immune antibody molecule to keep is removed the host's of immunization immunoglobulin or its analog from antibody.

This aspect of the present invention relates to the operation of non-human antibody's framework region.

The present invention extends to mutant, analog and the derivant that still keeps the specific main body antibody of La.

Term " mutant " or " derivant " comprise one or several aminoacid replacement, add and/or disappearance.

According to used herein, term " CDR " comprises three light chains in the variable part of antibody framework region of the bridge that exists on the bound fraction that covers molecule and the CDR structure ring in three heavy chain districts.These rings have characteristic typical structure (Chothia etc., J Mol.Biol.196:901,1987; Chothia etc., J.Mol.Biol.227:799,1992).

So-called " framework region " meaning is light chain immunoglobulin or variable region of heavy chain, wherein inserts three hypervariable regions, is also referred to as CDRs.The zone of accurately definite framework region and CDRs (referring to, for example, Kabat etc., " Sequences of Proteins of ImmunologicalInterest ", U.S.Department of Health and Human Services, 1983).The framework region sequence of different light chains or heavy chain is conservative relatively in a kind of species.According to used herein, " people's framework region " is the framework region (about 85% or higher, common 90-95% or higher) substantially the same with naturally occurring human normal immunoglobulin's framework region.The antibody framework region is the associating framework region that constitutes light chain and heavy chain, with deciding and arrange CDRs.CDRs mainly is responsible for combining with the La epi-position.

According to used herein, term " variable region of heavy chain " meaning is the polypeptide that length has about 110 to 125 amino acid residues, and its aminoacid sequence is corresponding to the aminoacid sequence from the heavy chain of the initial monoclonal antibody of the present invention of amino terminal (N-end) amino acid residue of heavy chain.Equally, term " variable region of light chain " meaning is the polypeptide that length has about 95 to 130 amino acid residues, and its aminoacid sequence is corresponding to the aminoacid sequence from the light chain of the initial monoclonal antibody of the present invention of amino terminal (N-end) amino acid residue of light chain.NH ₂-the variable region gene of terminal (about 110 aminoacid) and the κ of COOH-end or λ constant region gene code total length immunoglobulin " light chain " (approximately 25Kd or 214 aminoacid).Similarly, in variable region gene (about 116 aminoacid) and other above-mentioned constant region genes one, for example γ (about 330 aminoacid of encoding) coding total length immunoglobulin " heavy chain " (approximately 50Kd or 446 aminoacid).

Term " immunoglobulin " or " antibody " are used to refer to the protein that is made of one or several polypeptide of being encoded by immunoglobulin gene in fact here.The immunoglobulin gene of identification comprises κ, λ, alpha, gamma (IgG ₁, IgG ₂, IgG ₃, IgG ₄), δ, ε and μ constant region gene, and countless immune globulin variable region gene.A kind of form of immunoglobulin constitutes the base structure unit of antibody.This form is the tetramer, and by two identical immunoglobulin chains to constituting, each is to having a light chain and a heavy chain.Each centering, light chain and variable region of heavy chain are responsible for conjugated antigen together, and constant region is responsible for the antibody mediated effect subfunction.Except antibody, immunoglobulin can exist with various other forms, comprise, for example, Fv, Fab, Fab ' and (Fab ') ₂

The invention still further relates to segmental application and generation, comprise by the monoclonal antibody of method preparation of the present invention, for example, Fv, Fab, Fab ' and F (ab ') ₂Fragment.Such fragment can be by standard method, and for example (1991-1997, the above) method of Miao Shuing prepare Coligan etc.

The invention still further relates to have with monoclonal antibody of the present invention same or similar specific synthetic or the reorganization antigen-binding molecule.Such antigen-binding molecule can comprise the Fv fragment of synthesizing stableization.Such illustrative fragment comprises strand Fv fragment (sFv is often referred to as scFv), wherein uses peptide linker to make V _HThe N-terminal of domain or C-terminal respectively with V _LThe C-terminal of domain or N-terminal bridge joint.ScFv does not have all constant portions of whole antibody, and can not activating complement.Be used to connect V _HAnd V _LThe suitable peptide linker of domain is to make V _HAnd V _LDomain is folded into the polypeptide strand of the antigen binding site with three dimensional structure similar to the antigen binding site that derives the segmental whole antibody of Fv.By US Patent No 4,946,778 disclosed methods can obtain to have the joint of desirable properties.Yet, there is not joint in some cases, for example, can prepare ScFvs according to (Krebber etc., J.Immunol.Methods 201 (1) .35-55,1997) disclosed methods such as Krebber.Perhaps, can pass through US Patent No 5,091,513, European patent No 239,400 or Winter and Milstein (Winter and Milstein, Nature 349:293,1991) and the method described of the article of Plckthun etc. (Plckthun etc., In Antibody engineering.Apractical approach 203-252,1996) prepare them.

Perhaps, synthetic stable Fv fragment comprises the stable Fv of disulphide (dsFv), and wherein cysteine residues imports V _HAnd V _LDomain makes in complete folding Fv molecule, forms disulfide bond between two residues.For example (Glockshuber etc., Biochem.29:1363-1367,1990; Reiter etc., Biochem.33:5451-5459,1994; Reiter etc., Cancer Res.54:2714-2718,1994; Reiter etc., J.Biol.Chem.269:18327-18331,1994; Webber etc., Mol.Immunol.32.249-258,1995) in the appropriate method of preparation dsFv has been described.

Synthetic or the recombinant antigen-binding molecule that also relates to is a single variable region domain (being called dAbs), for example, is disclosed in (Ward etc., Nature 341.544-546,1989; Hamers-Casterman etc., Nature 363.-446-448,1993; Davies ﹠amp; Riechmann, FEBS Lett.339.285-290,1994).

Perhaps, synthetic or recombinant antigen-binding molecule can comprise " corpusculum (minibody) ".In this, corpusculum is the little modification of whole antibody, the strand primary element of its coding whole antibody.Suitably, antibody is by the V of the natural antibody that merges with the stranded district of immunoglobulin molecules and CH3 domain _HAnd V _LDomain constitutes, and is for example, as US Patent No 5,837, disclosed in 821.

In another embodiment, synthetic or recombinant antigen-binding molecule can comprise the deutero-protein framework of NIg.For example, can be with reference to (Ku ﹠amp; Schutz, Proc.Natl.Acad.Sci.USA92.6552-6556,1995), the document discloses to be had two and produces the four-helix bundle albuminous cell pigment b562 that CDR s is selected to the bonded ring of antigen at random.

Synthetic or recombinant antigen-binding molecule can be polyvalent (promptly having the antigen binding site more than).Such multivalent molecule can be specific for one or several antigen.Such multivalent molecule be can prepare by two antibody fragments by the peptide dimerization that contains cysteine, (Adams etc., Cancer Res.53:4026-4034,1993 for example, are disclosed in; Cumber etc., J.Immunol.149:120-126,1992).Perhaps, by making antibody fragment and naturally occurring dimer amphipathic helix merge (Plunckthun, Biochem.31:1579-1584,1992) or by using (the Kostelny etc. of preferential assorted dimerization, J.Immunol.148:1547-1553,1992) domain (for example leucine zipper jun and fos) can promote heterodimerization.

The invention further relates to amino acid whose chemical analog in the main body antibody.For example if desired to experimenter's dispenser, the application of amino acid whose chemical analog is useful especially for stable molecule.Here the amino acid whose analog that relates to includes but not limited to, the modification of side chain, peptide, the insertion of alpha-non-natural amino acid and/or their derivant in polypeptide or the protein synthesis, and the cross-linking agent and the use of protein molecule or their analog being forced the additive method of conformation constraint.

The example that the side chain that the present invention relates to is modified comprises for example by then using NaBH with aldehyde reaction ₄Reductive reductive alkylation is to the modification of amino; Amidine with the methyl acetimide turns usefulness into; Use the acetylation of acetic anhydride; Use cyanate that the carbamyl of amino is turned into usefulness; Use 2,4,6-trinitro-benzene-sulfonic acid (TNBS) is to the trinitro-benzylation of amino; Use succinic anhydrides and tetrahydrophthalic anhydride acylation to amino; Then use NaBH after acting on the trembling of pyrrole (pyridoxylation) of using the pyridoxal 5-phosphate ester to lysine ₄Reduction.

Use resembles 2, the 3-diacetyl, and the such reagent of phenylglyoxal and Biformyl can be modified the guanidine radicals of arginine residues by forming the heterocycle condensation product.

For example form corresponding amide and can modify carboxyl by then carrying out derivatization after the carbodiimides activation that forms through O-acyl group isourea.Can modify the Sulphydryl group by the method for example: the carboxymethylation that uses iodo acetic acid or iodo-acetamide; The oxidation performic acid is to cysteic acid; Mix two sulfur things with other mercaptan compounds formation; Maleimide reaction with maleimide, maleic anhydride or other replacements; Use 4-chlorine mercuri benzoate, 4-chlorine mercuri benzenesulfonic acid, phenyl mercury chloride, 2-chlorine mercuri-4-nitrophenol and other mercurial to form the hydrargyrum derivant; Carbamyl with cyanate under alkaline pH turns usefulness into.

By, for example, can modify trp residue to the alkylating of indole ring with the Oxidation of N-bromine butanimide or with 2-hydroxyl-5-nitrobenzyl bromine or sulphenyl halide.On the other hand, can form 3-nitrotyrosine derivant by nitration effect and modify tyrosine residue with tetranitromethane.

By carrying out alkylating with the iodoacetic acid derivant or carrying out the N-carbethoxylation with coke acid diethyl ester and can realize modification to the histidine residues imidazole ring.

The example that inserts alpha-non-natural amino acid and derivant in peptide is synthetic includes but not limited to, use nor-leucine, 4-aminobutyric acid, 4-amino-3-hydroxyl-5-phenylpentanoic acid, 6-aminocaprolc acid, tert-butyl group glycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or amino acid whose D-isomer.Table 2 provides the catalogue of the alpha-non-natural amino acid that relates to here.

Table 2

Non-common amino acid	Code	Non-common amino acid	Code
				Butyrine	Abu	The L-N-methylalanine	Nmala
Alpha-amido-alpha-methyl butyric acid ester	Mgabu	The L-N-methylarginine	Nmarg
				The 1-aminocyclopropane-1-carboxylic acid ester	Cpro	The L-N-methylasparagine	Nmasn
		The L-N-methylaspartic acid	Nmasp
				Aminoisobutyric acid	Aib	L-N-methyl cysteine	Nmcys
Amino norborny carboxylate	Norb	L-N-methyl glutamine	Nmgln
						L-N-methyl glutamic acid	Nmglu
Cyclohexylalanine	Chexa	The L-N-methylhistidin	Nmhis
				The cyclopenta alanine	Cpen	L-N-methyl isoleucine	Nmile
The D-alanine	Dal	The L-N-methylleucine	Nmleu
				The D-arginine	Darg	The L-N-methyllysine	Nmlys
The D-aspartic acid	Dasp	The L-N-methylmethionine	Nmmet
				The D-cysteine	Dcys	L-N-methyl nor-leucine	Nmnle
The D-glutamine	Dgln	L-N-methyl norvaline	Nmnva
				D-glutamic acid	Dglu	L-N-methyl ornithine	Nmorn
The D-histidine	Dhis	L-N-methylbenzene alanine	Nmphe
				The D-isoleucine	Dile	The L-N-methylproline	Nmpro
The D-leucine	Dleu	L-N-methyl serine	Nmser
				D-lysine	Dlys	The L-N-methylthreonine	Nmthr
The D-methionine	Dmet	The L-N-methyl tryptophan	Nmtrp
				The D-ornithine	Dorn	The L-N-methyl-tyrosine	Nmtyr
The D-phenylalanine	Dphe	The L-N-methylvaline	Nmval
				The D-proline	Dpro	L-N-Methylethyl glycine	Nmetg
The D-serine	Dser	L-N-methyl tertbutyl glycine	Nmtbug

The D-threonine	Dthr	The L-nor-leucine	Nle
				The D-tryptophan	Dtrp	The L-norvaline	Nva
D-tyrosine	Dtyr	Alpha-Methyl-aminoisobutyric acid esters	Maib
				The D-valine	Dval	Alpha-Methyl-γ-An Jidingsuan ester	Mgabu
D-Alpha-Methyl alanine	Dmala	The Alpha-Methyl Cyclohexylalanine	Mchexa
				D-Alpha-Methyl arginine	Dmarg	Alpha-Methyl cyclopenta alanine	Mcpen
D-Alpha-Methyl agedoite	Dmasn	Alpha-Methyl-Alpha-Naphthyl alanine	Manap
				D-Alpha-Methyl aspartic acid	Dmasp	The Alpha-Methyl penicillamine	Mpen
D-Alpha-Methyl cysteine	Dmcys	N-(the amino butyl of 4-) glycine	Nglu
				D-Alpha-Methyl glutamine	Dmgln	N-(2-amino-ethyl) glycine	Naeg
D-Alpha-Methyl histidine	Dmhis	N-(3-aminopropyl) glycine	Norn
				D-Alpha-Methyl isoleucine	Dmile	N-amino-alpha-methyl butyric acid ester	Nmaabu
D-Alpha-Methyl leucine	Dmleu	The Alpha-Naphthyl alanine	Anap
				D-Alpha-Methyl lysine	Dmlys	N-benzyl glycine	Nphe
D-Alpha-Methyl methionine	Dmmet	N-(2-carbamyl ethyl) glycine	Ngln
				D-Alpha-Methyl ornithine	Dmorn	N-(carbamoyl methyl) glycine	Nasn
D-Alpha-Methyl phenylalanine	Dmphe	N-(2-carboxyethyl) glycine	Nglu
				D-Alpha-Methyl proline	Dmpro	N-(carboxymethyl) glycine	Nasp
D-Alpha-Methyl serine	Dmser	N-cyclobutyl glycine	Ncbut
				D-Alpha-Methyl threonine	Dmthr	N-suberyl glycine	Nchep
D-Alpha-Methyl tryptophan	Dmtrp	The N-Cyclohexylglycine	Nchex
				The D-alpha-methyltyrosine	Dmty	N-ring decyl glycine	Ncdec
D-Alpha-Methyl valine	Dmval	N-cyclo-dodecyl glycine	Ncdod
				The D-N-methylalanine	Dnmala	N-ring octyl group glycine	Ncoct
The D-N-methylarginine	Dnmarg	N-cyclopropyl glycine	Ncpro
				The D-N-methylasparagine	Dnmasn	N-ring undecyl glycine	Ncund
The D-N-methylaspartic acid	Dnmasp	N-(2, the 2-diphenyl-ethyl) glycine	Nbhm
				D-N-methyl cysteine	Dnmcys	N-(3, the 3-diphenyl propyl) glycine	Nbhe
D-N-methyl glutamine	Dnmgln	N-(3-guanidine radicals propyl group) glycine	Narg
				D-N-methyl glutamic acid	Dnmglu	N-(1-ethoxy) glycine	Nthr
The D-N-methylhistidin	Dnmhis	N-(ethoxy) glycine	Nser
				D-N-methyl isoleucine	Dnmile	N-(imidazole radicals ethyl) glycine	Nhis
The D-N-methylleucine	Dnmleu	N-(3-indyl ethyl) glycine	Nhtrp
				The D-N-methyllysine	Dnmlys	N-methyl-γ-An Jidingsuan ester	Nmgabu
N-methylcyclohexyl alanine	Nmchexa	The D-N-methylmethionine	Dnmmet
				D-N-methyl ornithine	Dnmorn	N-methylcyclopentyl alanine	Nmcpen
Sarcosine	Nala	D-N-methylbenzene alanine	Dnmphe
				N-methylamino isobutyrate	Nmaib	The D-N-methylproline	Dnmpro
N-(1-methyl-propyl) glycine	Nile	D-N-methyl serine	Dnmser

N-(2-methyl-propyl) glycine	Nleu	The D-N-methylthreonine	Dnmthr
				The D-N-methyl tryptophan	Dnmtrp	N-(1-Methylethyl) glycine	Nval
The D-N-methyl-tyrosine	Dnmtyr	N-methyl-Alpha-Naphthyl alanine	Nmanap
				The D-N-methylvaline	Dnmval	N-methyl penicillanate amine	Nmpen
γ-An Jidingsuan	Gabu	N-(right-hydroxy phenyl) glycine	Nhtyr
				L-tert-butyl group glycine	Tbug	N-(sulfidomethyl) glycine	Ncys
The L-ethyl glycine	Etg	Penicillamine	Pen
				The L-homophenylalanin	Hphe	L-Alpha-Methyl alanine	Mala
L-Alpha-Methyl arginine	Marg	L-Alpha-Methyl agedoite	Masn
				L-Alpha-Methyl aspartic acid	Masp	L-Alpha-Methyl-tert-butyl group glycine	Mtbug
L-Alpha-Methyl cysteine	Mcys	L-Methylethyl glycine	Metg
				L-Alpha-Methyl glutamine	Mgln	L-Alpha-Methyl glutamic acid	Mglu
L-Alpha-Methyl histidine	Mhis	L-Alpha-Methyl homophenylalanin	Mhphe
				L-Alpha-Methyl isoleucine	Mile	N-(2-methyl sulfenyl ethyl) glycine	Nmet
L-Alpha-Methyl leucine	Mleu	L-Alpha-Methyl lysine	Mlys
				L-Alpha-Methyl methionine	Mmet	L-Alpha-Methyl nor-leucine	Mnle
L-Alpha-Methyl norvaline	Mnva	L-Alpha-Methyl ornithine	Morn
				L-Alpha-Methyl phenylalanine	Mphe	L-Alpha-Methyl proline	Mpro
L-Alpha-Methyl serine	Mser	L-Alpha-Methyl threonine	Mthr
				L-Alpha-Methyl tryptophan	Mtrp	The L-alpha-methyltyrosine	Mtyr
L-Alpha-Methyl valine	Mval	L-N-methyl homophenylalanin	Nmhphe
				N-(N-(2, the 2-diphenyl-ethyl) carbamoyl methyl) glycine	Nnbhm	N-(N-(3, the 3-diphenyl propyl) carbamoyl methyl) glycine	Nnbhe
1-carboxyl-1-(2,2-diphenyl-ethyl amino) cyclopropane	Nmbc

Cross-linking agent can be used for, and for example, stablizes the 3D conformation, uses the homotype bi-functional cross-linking agent, for example has wherein n=1 to n=6 (CH ₂) _nThe difunctional polyurethane of spacer groups, glutaraldehyde, N-hydroxy-succinamide ester and special-shaped bifunctional reagent, described reagent comprises amino reactive moieties usually, for example N-hydroxy-succinamide and other group specificity reactive moieties, for example dimaleoyl imino or disulfide group part (SH) or carbodiimides (COOH).

The invention further relates to the analytic process to biology sample detection programmed cell death cell, described analytic process comprises following step :-(3) make at the reactivity molecule contact of karyon molecule or its antigenic determinant suspects the biological sample that contains described karyon molecule; With

(4) complex that forms in the step (1) is carried out signal detection step,

Wherein detecting non-karyon interacting molecule-karyon molecular complex formation is the indication of programmed cell death cell.

Preferably, described karyon molecule is La.

More preferably, described interacting molecule is an immunological molecule, is more preferably anti--La antibody, for example monoclonal antibody SW3 or 3B9.

Most preferably, described non--karyon location is born of the same parents' outside fixs, more preferably, be positioned in the kytoplasm of programmed cell death cell or in the plastidogenetic programmed cell death body of programmed cell death.

Signal detection step comprises that ELISA or any other reporter molecule are basic analytic process.As this part that detects step, at first need signal is enlarged.Should be appreciated that this analytic process can interior or external the carrying out of body.

For programmed cell death cells contacting onlooker-cell growth is hindered or onlooker-cell killing reagent, be cytostatics or kill cell reagent, of the present inventionly go immune monoclonal antibody A also can be used in the body programmed cell death imaging and be used for directed programmed cell death cell.

Anti--La antibody is better than present known product, for example Apomate ^TM(North AmericanScientific Inc.), is used for detecting in the body programmed cell death cell.Apomate ^TMUse the radio-labeled annexin V to come particularly to find out the kickback of cancer to chemotherapy in conjunction with the programmed cell death cell.Annexin V is in conjunction with Phosphatidylserine, its programmed cell death early stage ' flip-flops ' is to outer primary plasma membrane lobule.Yet, pass through Apomate ^TMThe detection of the tumor cell programmed cell death that chemotherapy is caused is variable because administration time to select be conclusive (Blankenberg etc., Clin Cancer Res 2002).Selection of time with use anti--that La antibody is compared importance is less, because antibody makes the programmed cell death cell directional in the macrophage that gathers at the cancer position.In addition, annexin V suppresses the phagocytosis of the macrophage-mediation of external programmed cell death thymocyte cell, surround it by the receptor-mediated absorption of the erythrocytic Fc of antibodies, it has the Phosphatidylserine (Callahan etc. of surface-exposure, Cell Death Different 7 (7): 645-653,2000; Cell Death Different 6 (2): 183-189 such as Krahlung, 1999).Therefore, anti--La antibody is nursed one's health the programmed cell death cell and is helped macrophage they is carried out phagocytosis.Especially, make macrophage be oriented to diagnosis and/or therapeutic purposes, as hereinafter in greater detail.Therefore, relatively for example to detect those present known diagnostic methods of annexin V, one of advantage of using anti--La antibody is: macrophage causes the accumulation of antibody antibody in the macrophage at programmed cell death cell position to the conditioning and the absorption of programmed cell death cell, thereby helps imaging.

About imaging, reporter molecule with go immune monoclonal antibody to connect together, import the host then, for example the people.By detecting reporter molecule, can visible cell programmed cell death bunch, for example relevant those with tumor.A kind of useful especially reporter molecule form is the karyon labelling.Some radiosiotope allows by Positron Emission Tomography (PET) imaging, and some parts help detecting the target combination by nuclear magnetic resonance (MRI).

Therefore, another aspect of the present invention relates to the method to people detection programmed cell death cell, described method comprises to described patient introduces the interacting molecule at karyon molecule or its antigenic determinant of using the reporter molecule labelling, the selection portion branch of interacting molecule by blood circulation or blood circulation by labelling disseminated, then described patient is used the reporter molecule detection method to identify the location of interacting molecule.

Preferably, described karyon molecule is La.

More preferably, described interacting molecule is an immunological molecule, is more preferably anti--La antibody.

Most preferably, described non--karyon location is born of the same parents' outside fixs, preferably, be positioned in the kytoplasm of programmed cell death cell or in the plastidogenetic programmed cell death body of programmed cell death.

Preferably, described programmed cell death cell is the tumor feature.

Preferably, described tumor cell is central nervous system's tumor, retinoblastoma, neuroblastoma and other department of pediatrics tumors, head and neck cancer (for example squamous cell carcinoma), breast and carcinoma of prostate, pulmonary carcinoma (small cell lung cancer and nonsmall-cell lung cancer), renal carcinoma (for example renal cell adenocarcinoma), esophagus gastric cancer, hepatocarcinoma, the ductus pancreaticus biloma forms (for example adenocarcinoma pancreatic endocrine tumor), colorectal carcinoma, cervical cancer and anus cancer, uterus and other reproductive tract cancers, carcinoma of urethra (for example ureter and bladder cancer), blastocyte tumor (for example testis blastocyte tumor or ovary blastocyte tumor), ovarian cancer (for example epithelial ovarian cancer), unknown primary carcinoma, human immune deficiency associated malignancies (for example Kaposi ' s sarcoma), lymphoma, leukemia, malignant melanoma, sarcoma, endocrine tumor (for example thyroid tumor), mesothelioma and other pleural neoplasms, one of neuroendocrine tumor and cancer sample tumor feature.

Immune-base La detection method can be used various forms.For example, each comprises that at specific antigen or cancerous cell La has not homospecific multiple antibody and fixes in array.Making then from bioptic cells contacting antibody array and with the fixed cell is that tumor type is diagnosed out on the basis.

Also can carry out other more common analytic process, for example by ELISA, western blot analysis, immunoprecipitation analysis, immunofluorescence analysis, immunochemical analyses or facs analysis.

Therefore, the invention provides method to sample detection La or its fragment, variant or derivant, comprise and make sample contact antibody or its fragment or derivant, and detect the complex that comprises described antibody and La or its fragment, variant or derivant, wherein the non-karyon location of La is the indication of programmed cell death.

Preferably, described non-karyon location is born of the same parents' outside fixs, preferably, is positioned in the kytoplasm of programmed cell death cell or in the plastidogenetic programmed cell death body of programmed cell death.

As discussed above, can use and measure any suitable technique that complex forms.For example, can in immunoassay, use and the associating antibody of the present invention of reporter molecule.Such immunoassay includes but not limited to radio immunoassay (RIAs), enzyme linked immunological absorption method of testing (ELISAs) and immune chromatograph technology (ICTs), and Western blotting, these all well known to a person skilled in the art.For example, can be with reference to " Current Protocols in Immunology ", 1994, it discloses can be according to the various immunoassays of use of the present invention.Immunoassay comprises the competition method of testing.Should be appreciated that and the present invention includes qualitative and quantitative immunoassay.

For example United States Patent(USP) Nos. 4,016, described suitable immunoassay technology in 043,4,424,279 and 4,018,653.These comprise non-competing type unit point and the test of two-site, and the tradition competition is in conjunction with method of testing.These methods of testing comprise that also antigen-the binding molecule of guide markings combines with target antigen, and described in this case antigen is La or its fragment.

Two-site method of testing is useful especially in use of the present invention.Various methods of testing are arranged, and these all are included in the present invention.In brief, in typical early stage method of testing, do not have the antigen-binding molecule of labelling for example not have the antibody of labelling to be fixed on the solid substrate, and the sample of analyzing is contacted bonded molecule.Being incubated after the suitable time, allowing antibody-antigenic complex form enough a period of times, add another kind of antigen-binding molecule then, is anti-to antigen specific suitable two with the reporter molecule labelling that can produce detectable signal.Responseless material is all washed, and measure antigenic existence by the signal of observing the reporter molecule generation.Obtain qualitative results by simple observation optical signal, perhaps by the acquisition quantitative result of comparing with the antigenic control sample that contains known quantity.The change of previous method of testing comprises method of testing simultaneously, wherein adds the antibody of sample and labelling simultaneously to bonded antibody.These technology are well known to a person skilled in the art, comprise the little change that manifests easily.

In typical early stage test, have specific one to resist and surface of solids covalency or passive the combination to antigen or its antigen part.The described surface of solids is glass or polymer typically, and the polymer of normal use is a cellulose, polyacrylamide, nylon, polystyrene, polrvinyl chloride or polypropylene.Solid carrier can be pipe, bead, microplate, perhaps is fit to carry out any other surface of immunoassay.Associated methods is well known in the art, and generally comprises crosslinked covalent bond or physical absorption, is being used for the preparation washing copolymer-antibody complex of analytic sample.Add sample equal portions and the enough a period of times of incubation that to analyze to the solid phase complex then, allow antigen and the antibodies that exists.Incubation is after a period of time, the washing antigen-antibody complexes and dry and with have specific two temperature resistances for an antigenic part and educate.Two anti-generally associations with reporter molecule are used to refer to two and anti-ly combine with antigenic.According to associating reporter assay, the amount of the antibody of bonded labelling and proportional with immobilization one anti-bonded antigenic amount.

Another kind method relates to immobilized antigen in biological sample, allow then fixed antigen contact specific antibody, described antibody can be with the reporter molecule labelling or do not have a labelling.According to the amount and the reporter molecule signal intensity of target thing, by detecting bonded antigen with the direct labelling of antibody.Perhaps, allow two of an anti-specific labelling is resisted the anti-complexs of contact target things-, form anti--two anti-ternary complexs of target thing-.Signal detection complex by the reporter molecule emission.

As mentioned above, be appreciated that with the associating reporter molecule of antigen-binding molecule can comprise following:

(a) reporter molecule directly is connected with antibody;

(b) reporter molecule is connected with antibody indirect; That is, reporter molecule is connected with another kind of analytical reagent, and it is followed and antibodies; With

(c) be connected with the product subsequently of antibody.

Reporter molecule can be below comprising be selected the group of material: chromogen, and catalyst, enzyme, fluorescent dye, chemiluminescent molecule, paramagnetic ion, lanthanide ion be europium (Eu for example ³⁴), radiosiotope comprises other karyon labels, semiconductor-quantum-point (Wu etc., NatureBiotechnol 2002) and direct visual marking.Can prepare recombinant antibodies-sample molecule by for example strengthening green fluorescent protein (EGFP) fusion with counter pair.

Under direct visual marking situation, can use colloidal metal or non-metallic particle, dye granule, enzyme or substrate, organic polymer, latex particle, liposome, perhaps other contain the vesicle of the material that produces signal etc.

United States Patent(USP) Nos. U.S.4,366,241, U.S.4,843,000, and U.S.4,849,338 have described the enzyme that is suitable as reporter molecule in a large number.The suitable enzyme that uses among the present invention comprises alkali phosphatase, horseradish peroxidase, luciferase, beta galactosidase, glucoseoxidase, lysozyme, malic dehydrogenase or the like.Enzyme can use separately or with solution in second kind of enzyme unite use.

Suitable fluorescent dye includes but not limited to, fluorescein isothiocyanate (FITC), and tetramethylrhodamin isothiocyanate (TRITC), R-phycoerythrin (RPE) and Texas are red.

Other fluorescent dyes of giving an example comprise those of following documents: Dower etc., international open No.WO 93/06121.Also can be with reference to the fluorescent dye of U.S. Patent No.s such as U.S. Patent No.s such as Singer 5,573,909 and Brinkley 5,326,692 descriptions.Perhaps, can be with reference to United States Patent(USP) Nos. 5,227,487,5,274,113,5,405,975,5,433,896,5,442,045,5,451,663,5,453,517,5,459,276,5,516,864,5,648,270 and 5,723,218 fluorescent dyes of describing.

Under enzyme immunity test situation, enzyme and two anti-couplings, general using glutaraldehyde or periodate.Yet, understand the various different coupling technologies that capable field technique personnel obtain easily easily.The general selected detectable change color that produces when coming of the substrate that has enzyme-specific that uses by corresponding enzyme hydrolysis.The example of suitable enzyme comprise above-described those.Can also use fluorescence substrate, obtain fluorescence-causing substance except product color substrate above-mentioned.In all cases, enzyme-traget antibody adds to an anti--antigenic complex, makes combination, washs excessive reagent then.The solution that will contain suitable substrates then adds to antibody-antigen-antibody complexes.Substrate with the two anti-enzyme reactions that are connected, provide qualitative optical signal, it is quantification further, normally spectrophotometric method provides the indication of the antigenic amount that exists in the sample.

Perhaps, fluorescent chemicals, fluorescein for example, rhodamine and group of the lanthanides, europium (EU) can not change their binding ability with the antibody chemical coupling.When activating by the specific wavelength optical illumination, the antibody absorption luminous energy of fluorescent dye-labelling brings out the molecule stress state, then sends the light that can estimate the characteristic color of detection with optical microscope.Allow fluorescence-traget antibody in conjunction with an anti--antigenic complex.Washing off does not have after the bonded reagent, then residual ternary complex is exposed to the light of suitable wavelength.Observed fluorescence is indicated interested antigenic existence.Immunofluorescence assay (IFMA) in this area be determine and be used in particular for the inventive method.Yet, also can use other reporter molecules, for example radiosiotope, chemiluminescent substance or bioluminescent molecules.

Method of the present invention is as thinking that it is that of those dangerous individualities of the development of feature or disease (for example tumor) finishes test or as the monitor of development with the cell programmed cell death that generation is arranged, perhaps as at inhibition or slow down a kind of like this monitor of therapeutic or prophylactic treatment effect of disease process in addition.In these cases, with the adjusting mapping of La level in a class or a few class biological sample, be individual state or the therapeutic of using at present or the valuable index of prophylactic treatment effect.Therefore, should be appreciated that method of the present invention extends to individuality with respect to their normal levels (defining as mentioned) or with respect to the label level rising of one or more label levels early of measuring from the biological sample of described individuality or the monitoring of reduction.

The invention further relates to purposes at interacting molecule quantitative or sxemiquantitative diagnostic kit of programmed cell death cell in preparation detects from patient's biological sample of karyon molecule.Test kit can have operation instructions, and can be form automatic or automanual or that be complementary with automaton or software.

Preferably, described karyon molecule is La.

Preferably, described programmed cell death cell is the programmed cell death tumor cell.

The application of where face restriction test kit not in office is used for using in diagnosis and research and detects the programmed cell death cell.At present, the reagent that a lot of vitro detection programmed cell deaths are arranged on the market, comprise annexin V, mitochondrion permeability dyestuff, APO2.7 (the only monoclonal antibody of identification mitochondrial protein during programmed cell death), DNA binding fluorescent dyes, for example iodate third ingot and 7-aminoactinomycin D (7-AAD) and fluorescence caspase inhibitor.Yet, these reagent can not be distinguished programmed cell death cell and non-viable non-apoptotic cell (H.Lecoeur etc., and need more than one to come specificity to identify late programmed cell death cell (P.Smolewski etc., J.Immunol Methods2002 J Immunol Methods 2002); Hamel etc., Cytometry25 (2): 173-181,1996).

According to the present invention, the generation of Anti La antibody is at activity or inactive form molecule.

Except the unquestionable diagnosis benefit of the inventive method, accurately the ability of targeting programmed cell death cell provides now with location and sending of high orientation mode and passs therapeutic and/or method of prophylactic treatment.Up to now, such treatment (often being called " magic power bullet ") is still impossible.Especially, with regard to oncotherapy since can not identify antibody at the fact of suitable tumor specific antigen, the targeted therapy notion still is impossible.Yet the present invention implements therapeutic or prophylactic treatment by orientation to the programmed cell death cell that comprises experimenter's tumor, has overcome these shortcomings.By select can with anti--La immunological molecule coupling, but to being positioned at therapeutic or preventative effector mechanism, can realize effectively killing and wounding of tumor near the cell performance function of the non-programmed cell death tumor cell of programmed cell death cell.Experimenter's effector mechanism can be taked any suitable form, but preferably send the non-programmed cell death tumor cell of passing toxicity molecule or killing close position in addition.

In addition, resemble the phagocytosis that the such antibody of human IgG I and IgG3 helps using anti--programmed cell death cell that La antibody was nursed one's health.Do not limit the invention to any theory or model of action, anti--La antibody becomes and concentrates one's gaze on and accumulate in macrophage in the body or other have in the phagocytotic cell.The cell of experience programmed cell death at first divides film forming-bound fraction or programmed cell death body, its then by around cell, particularly by known be that the professional scavenger cell of macrophage is handled.The programmed cell death cell of moment in the external and body internal specific Recognition and Programming cell death process of anti--La antibody.In addition, be positioned at macrophage in anti--preferential body of La antibody, it engulfs the programmed cell death cell.Macrophage is to heart attack, and the healing of the tissue injury that takes place in apoplexy and the organ transplantation rejection has contribution.Cancer has the high-load macrophage that is known as tumor-associated macrophages, and it can postpone or promote the growth of cancer.Therefore, anti--La antibody conduct is to being used to send the carrier of passing the therapeutic activity technology.

Therefore, it is the method for the disease of feature with the cell programmed cell death that another aspect of the present invention relates to experimenter's therapeutic and/or prophylactic treatment, described method is included in to be enough to treat under the time and condition of described disease, described experimenter is used the interacting molecule at karyon molecule or its antigen fragment of effective dose, and described interacting molecule is connected with therapeutic or preventative effector structure, bonding or associate in addition.

Preferably, described karyon molecule is La.

More preferably, described interacting molecule is an immunological molecule, is more preferably anti--La antibody.

In another preferred embodiment, described disease is cardiac muscle or cerebral tissue infraction, autoimmune and other inflammation diseases, virus disease, AIDS for example, neurodegenerative disease, for example presenile dementia or parkinson disease, acute solid organ or bone marrow transplantation rejection, tissue injury that chemotherapy or radiotherapy cause (' mucositis ') or tumor are as tumor.

Tumor and tumor cell involved in the present invention include but not limited to, central nervous system's tumor, retinoblastoma, neuroblastoma and other department of pediatrics tumors, head and neck cancer (for example squamous cell carcinoma), breast and carcinoma of prostate, pulmonary carcinoma (small cell lung cancer and nonsmall-cell lung cancer), renal carcinoma (for example renal cell adenocarcinoma), esophagus gastric cancer, hepatocarcinoma, the ductus pancreaticus biloma forms (for example adenocarcinoma pancreatic endocrine tumor), colorectal carcinoma, cervical cancer and anus cancer, uterus and other reproductive tract cancers, carcinoma of urethra (for example ureter and bladder cancer), blastocyte tumor (for example testis blastocyte tumor or ovary blastocyte tumor), ovarian cancer (for example epithelial ovarian cancer), unknown primary carcinoma, human immune deficiency associated malignancies (for example Kaposi ' s sarcoma), lymphoma, leukemia, malignant melanoma, sarcoma, endocrine tumor (for example thyroid tumor), mesothelioma and other pleural neoplasms, neuroendocrine tumor and cancer sample tumor.

About " karyon molecule ", " La ", " immunological molecule ", " experimenter " should be appreciated that to have and identical meaning above-mentioned with " programmed cell death ".

The more special method that provides experimenter's therapeutic and/or prophylactic treatment tumor disease of the present invention, described method be included in be enough to suppress, reduce or in addition decrement regulate under the time and condition of tumor growth, described experimenter is used the immunological molecule at La or its antigen part of effective dose, and described immunological molecule is connected with therapeutic or preventative effector structure, bonding or associate in addition.

Preferably, described tumor disease is a malignant tumor.

Be construed as the disease that is meant direct or indirect treated tissue when being arranged in programmed cell death cell site, any suitable structure of for example decrement regulate tumor cell growth about " effector structure ".In this embodiment preferred, the effector structure most likely realizes this result's the protein or the group of nonprotein molecule or molecule.The effector example of structure that is fit to use in the methods of the invention includes but not limited to:

(i) with other factors of one or several aspect that cytokine, chemotactic factor or performance are induced or enhance immunity is replied, for example macrophage, dendritic cell and/or t cell activation agent connect, thereby increase the application of the antibody that the onlooker kills and wounds.For example; chemotactic peptide; N-formoxyl-methionyl-leucyl-phenylalanine (FMLP) (Morikawa etc.; CancerImmunol Immunother 27 (1): 1-6; 1988) and new antibacterial lipopeptid; JBT 2002 (Shinohara etc., J Immunother 23 (3): 321-331,2000) is two kinds of activator of tumor-associated macrophages.

(ii) with the application of toxin conjugated antibody.

Should be appreciated that any suitable protein or the nonprotein molecule that is meant the purpose that realizes providing the signal that reduces, prevents or suppress experimenter's cell proliferation in addition, break up or keep (being called " decrement is regulated growth " of described cell here) about " toxin ".Described toxin can work by various methods, and comprising by directly contacting with experimenter's cell provides signal or emitting molecule or particle, for example radiates under radiosiotope toxin situation, provides signal to experimenter's cell.Preferably, toxin is a radiosiotope, is more preferably at short notice height toxicity and the short half-life is arranged, thereby make the contiguous on every side minimized radiosiotope of the involuntary toxic generation of non-target thing cell.The most especially, described radiosiotope is the radioisotopic alpha-particle of emission.Yet, should be appreciated that radiosiotope is not limited to the radioisotopic alpha-particle of emission, and can comprise β-and γ-emission radiosiotope according to clinical setting.The radioisotopic example of α-emission that is fit to use in the methods of the invention includes but not limited to Tb-149 or Bi-213.Should be appreciated that the toxin that uses in the methods of the invention can be purification, partial purification or the form that does not have purification.It also forms a more macromolecular composition.Toxin can be naturally occurring or the preparation of can synthesizing or recombinate.

Should be appreciated that other examples that drop on the molecule in " toxin " scope comprise Ricin, colicheamicin, prodrug (as antibody-directed prodrug invertase therapy [ADEPT]) and new Biotherapeutics material, for example catalytic antibody.

Should be appreciated that method of the present invention can interior or external the carrying out of body.External examples of applications includes but not limited to, contains the external removing of the biological sample of tumor cell.For example, take out bone marrow and/or blood from the patient, the method according to this invention is removed and the kill tumor cell, fails back then and gives the patient.Make at present in this way, but, avoid the relevant big risk of incompatible bone marrow transplantation with MHC with more unconspicuous oriented approach.

About with anti--La, for example the effector structure of antibody or other immunological molecules " connection, bonding or associate in addition " should be appreciated that and is meant the covalently or non-covalently interaction mechanism of realizing that two molecules connect.This includes but not limited to use peptide bond, ionic bond, hydrogen bond, Van der Waals force or any other interaction bonding mechanism.

Should be appreciated that about " growth " of cell or tumor the propagation, differentiation and/or the survival that are meant experimenter's cell keep, and propagation, differentiation and/or survival that " decrement is regulated growth " of cell or tumor is meant the cell ageing process or refers to reduce, prevent or suppress experimenter's cell are kept.In preferred embodiments, experimenter growth be that propagation and experimenter's decrement regulate is to kill.In this, send by pair cell and pass havoc or pair cell and send and pass inducing cell programmed cell death signal and can realize cell killing.

" treatment " is thought its broader terms about " therapeutic " or " preventative ".Term " treatment " hints that not necessarily the experimenter is treated recovery fully.Similarly, " preventative " must not refer to that the experimenter does not catch at last.Therefore, treatment and prevention comprise the danger that alleviates or prevent or reduce in addition to produce particular disorder of particular disorder sign.The severity of particular disorder or initial can be thought to reduce in term " prevention "." treatment " be the order of severity of the disease that can reduce to have existed also.

Do not limit the invention to any theory or binding mode, anticancer therapy kills by programmed cell death usually, but under a lot of terminal cancer situations, some cancerous cell opposings can be passed through the inductive programmed cell death of specific anticancer therapy.These programmed cell death-resisting cells are roots of disease clinical recurrence, kill most of patient and a big chunk early-stage cancer patient of terminal cancer at last.Those patient with advanced cancer show the initial modality of corresponding treatment, because the proof interior tumor cell kills and wounds, if also used another kind of non-cross tolerance treatment modalities, then can win survival and quality of life in addition.Therefore,, use the inventive method to diagnose, can identify those patients that benefit from therapeutic conjugate or the supplement therapy of heterozygosis fusion rotein the cancer patient who replys is arranged as what describe in detail.

Under auxiliary clinical scenarios, use to resist-La antibody also is useful.Though early stage breast and colon cancer can be cured by operation, but the risk that obvious and incurable whole body recurrence is arranged, because primary tumo(u)r has those patients that some excessive risk feature and/or regional nodes comprise transfer, may exist does not have detected whole body micrometastasis.Like this, a fraction of these patients are in addition cured in adjuvant chemotherapy and/or auxiliary hormone therapy (under the mastocarcinoma situation), and are general because successfully removed the whole body micrometastasis.

In addition, though because there is not blood supply dormancy tumor to keep little state, the tumor cell fast updating in the infringement, its cell division speed and programmed cell death velocity balance.Therefore, in addition the dormancy tumor also be the suitable target thing of the technology of the present invention.Under clinical obvious transfer and micrometastasis both of these case, programmed cell death-resistance cancerous cell can mix with susceptible cancerous cell.If to making near the cancerous cell of programmed cell death send non-cross tolerance and/or the Synergistic method of passing tumor-killing by treatment for the first time, these survival cancerous cell then can take place replenish and kill and wound.Help the other technology of this technology can improve its therapeutic effect to look on kill potential.

Therefore, the most preferred embodiment of the present invention relates to the treatment of metastatic carcinoma.

According to this preferred embodiment, method to experimenter's therapeutic treatment metastatic carcinoma is provided, described method be included in be enough to suppress, reduce or in addition decrement regulate under the time and condition of described metastatic carcinoma growth, described experimenter is used the immunological molecule at La or its antigen part of effective dose, and described immunological molecule is connected with the therapeutic effects minor structure, bonding or associate in addition.

As described in detail above, the present invention should also be appreciated that the decrement adjusting that extends to growth of tumour cell under the external environment.For example, can remove tumor cell, autograft then from bone marrow or the hepatocellular culture fluid of peripheral blood.

" effective dose " meaning is to obtain Expected Response to small part, perhaps postpones taking place or inhibition process or both all stagnate the generation of the particular disorder of being treated or develop necessary amount.This amount changes according to following factor: the health of individuality to be treated and health, and the sorted group of individuality to be treated, the desired protection degree, composite preparation, the medical science situation is estimated and other correlative factors.Expect that this amount drops within the wide relatively scope that can determine by routine test.

The invention further relates to therapeutic alliance, for example in treatment of cancer, use in the antibody of the present invention, make mammal accept circulating cells drug toxicity or radiotherapy.

Can implement the administration of the interacting molecule (being called " regulator " here) of pharmaceutical compositions by any method easily.When with according to the amount administration of particular case the time, the regulator of pharmaceutical composition shows therapeutic activity.Change and to depend on for example human or animal's and regulator selection.Can use wide region dosage.Consider the patient, can use the regulator of for example about 0.1mg every day to about 1mg for every kg body weight.Can regulate dosage regimen, optimum therapeutic response is provided.For example, can be continuously, every day, weekly, every month or other suitable intervals are with several dosed administrations that separate, perhaps the emergency according to its situation reduces dosage in proportion.

Can use regulator in a suitable manner, for example by oral, intravenous (under the water solublity situation), intraperitoneal, intramuscular, subcutaneous, intradermal or suppository approach or imbed (for example using the slow release molecule).Can use regulator with pharmacy acceptable nontoxic salt form, for example acid-addition salts or metal complex are for example with the salt or the metal complex (it thinks the salt for the application's purpose) of zinc, ferrum etc.The illustration of such acid-addition salts is a hydrochlorate, hydrobromate, sulfate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, Ascorbate, tartrate or the like.If the active component of using is a tablet form, tablet can contain binding agent, Tragacanth for example, corn starch or gelatin; Disintegrating agent, for example alginic acid; And lubricant, for example magnesium stearate.

Route of administration includes but not limited to, breathes, and in the trachea, nasopharynx, intravenous, intraperitoneal, subcutaneous, intracranial, intradermal, intramuscular, ophthalmic, in the brain, intranasal, input, oral, rectally is by IV instillation, patch and implant.

According to these methods, the medicine that limits according to the present invention can with one or more other chemical compounds or molecule co-administered.So-called " co-administered " meaning is in identical preparation or with two preparations that separate, by the administration simultaneously of identical or different approach, perhaps by identical or different approach administration successively.For example, for reinforced effects, medicine of the present invention can be with the agonist administration.So-called " successively " administration meaning be use between two types of molecules at interval several seconds, several minutes, a few hours or a couple of days interval.These molecules can be with any order administration.

The present invention relate on the other hand with the effector structure link coupled anti--karyon interaction of molecules molecule is used to prepare the purposes to the sanatory medicine of experimenter, described disease is a feature with the cell programmed cell death, and wherein said effector structure is treated described disease.

Preferably, described karyon molecule is La.

Preferably, described interacting molecule is an immunological molecule, more preferably anti--La antibody, for example monoclonal antibody.

Most preferably, described karyon location is born of the same parents' outside fixs, as top defined.

In a further preferred embodiment, described disease is cardiac muscle or cerebral tissue infraction, autoimmune and other inflammation diseases, virus disease, AIDS for example, neurodegenerative disease, for example presenile dementia or parkinson disease, acute solid organ or bone marrow transplantation rejection, tissue injury that chemotherapy or radiotherapy cause (' mucositis ') or tumor are as tumor.

The tumor that the present invention relates to and the example of tumor cell include but not limited to central nervous system's tumor, retinoblastoma, neuroblastoma and other department of pediatrics tumors, head and neck cancer (for example squamous cell carcinoma), breast and carcinoma of prostate, pulmonary carcinoma (small cell lung cancer and nonsmall-cell lung cancer), renal carcinoma (for example renal cell adenocarcinoma), esophagus gastric cancer, hepatocarcinoma, the ductus pancreaticus biloma forms (for example adenocarcinoma pancreatic endocrine tumor), colorectal carcinoma, cervical cancer and anus cancer, uterus and other reproductive tract cancers, carcinoma of urethra (for example ureter and bladder cancer), blastocyte tumor (for example testis blastocyte tumor or ovary blastocyte tumor), ovarian cancer (for example epithelial ovarian cancer), unknown primary carcinoma, human immune deficiency associated malignancies (for example Kaposi ' s sarcoma), lymphoma, leukemia, malignant melanoma, sarcoma, endocrine tumor (for example thyroid tumor), mesothelioma and other pleural neoplasms, neuroendocrine tumor and cancer sample tumor.

On the other hand, the present invention relates to contain the pharmaceutical composition of regulator defined above and one or more pharmaceutical acceptable carriers and/or diluent.Described regulator refers to active component.

The medicament forms that is fit to the injection use comprises aseptic aqueous solution (under the water solublity situation) or the dispersion and the sterilized powder agent of the interim preparation that is used for aseptic parenteral solution or dispersion, perhaps can be any form of cream forms or suitable local application.It must be stable under preparation and condition of storage and must for example contamination of antibacterial and fungus of reservation antimicrobial.Carrier can be to contain, water for example, ethanol, polyhydric alcohol (for example glycerol, propylene glycol and liquid macrogol etc.), its suitable mixture, and vegetable oil.By using for example lecithin of coating, under the dispersion situation by keeping needed granularity and by using surfactant can keep suitable flowability.By various antibacteriums and antifungal, p-Hydroxybenzoate for example, methaform, phenol, sorbic acid, thimerosal etc. can prevent microbial action.Under many circumstances, preferably include isotonic agent, for example, sugar or sodium chloride.Postpone absorbent by using in compositions, for example aluminum monostearate and gelatin can realize that the prolongation of Injectable composition absorbs.

As required, the reactive compound by in suitable solvent, adding necessary amounts and above various other compositions of exemplifying, then sterilising filtration prepares sterile injectable liquid.Generally speaking, by in the aseptic excipient that contains basic disperse medium and those other essential compositions that exemplify above, adding the various sterile actives dispersion that becomes to assign to prepare.Prepare under the sterilized powder agent situation of aseptic parenteral solution being used to, preferred manufacturing procedure is vacuum drying and freeze drying technology, obtains active component and adds powder agent from the composition of the other expectation of the solution of its prior aseptic filtration.

When active component during by due care, they can perhaps be sealed in hard or soft shell body gelatine capsule for example with inert diluent or with assimilating the edible carrier oral administration, perhaps can suppress in flakes, perhaps can directly mix with food.For the oral medication administration, reactive compound can use with mixed with excipients and can absorb forms such as tablet, buccal, lozenge, capsule, elixir, suspensoid, syrup, paper wafer.Such compositions and preparation should contain the reactive compound of at least 1% weight.Certainly, the percentage ratio of compositions and preparation can change, and aptly about 5% between about 80% unit of weight.The amount of reactive compound is for obtaining proper dosage in such therapeutic composition.Preparation is preferred compositions or preparation according to the present invention, makes oral dosage unit form contain about 0.1 microgram to the reactive compound between the 2000mg.

Tablet, lozenge, capsule etc. can also contain the composition of listing below: can add binding agent, and for example gummy, arabic gum, corn starch or gelatin; Excipient, for example dicalcium phosphate; Disintegrating agent, corn starch for example, potato starch, alginic acid or the like; Lubricant, for example magnesium stearate; And sweeting agent, sucrose for example, lactose or glucide, perhaps correctives, Herba Menthae for example, oil of wintergreen, or Fructus Pruni pseudocerasi flavouring agent.When dosage unit form is a capsule, it can contain liquid-carrier except top types of material.Can there be various other materials, as coating or change the physical form of dosage unit in addition.For example, tablet, pill or capsule can come coating with Lac sugar or both.Syrup or elixir can contain reactive compound, as the sucrose of sweeting agent, and as the methyl parahydroxybenzoate and the propyl p-hydroxybenzoate of antiseptic, dyestuff and correctives, for example Fructus Pruni pseudocerasi or Fructus Citri junoris flavouring agent.Certainly, any material that uses in the unit dosage form in preparation should be that pharmacy is pure and be nontoxic basically during in use amount.In addition, reactive compound can join in slow release goods and the preparation.

Also have, another aspect of the present invention relates to the medicine that ought use in the methods of the invention defined above.

Further specify the present invention by following non-limiting example.

Embodiment 1

Used herein resisting-La antibody is mouse monoclonal antibody hybridoma 3B9, and it discerns people and the mice form of small nuclear ribonucleoprotein antigen La/SS-B.Antibody sources is in Tom Gordon professor, Flinders Medical Centre, Adelaide, South Australia, he is from Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, doctor M.Bachmann of USA obtains there.The working group of Dr.Gordon discloses 3B9 (2002a such as Tran).Corresponding isotype control antibodies hybridoma is Sal5.

To human serum (La serum) intravenous injection that six wild types (ST) C57BL/6 male mice (seven week age) or two mice prostate transgenic adenocarcinoma in age in June (TRAMP) mice (the 1st hurdle) every day gives 400 microlitre normal human serums (NHS) or contain La-reaction autoantibody for twice.Injecting for the first time the complete or operation castrating (the 2nd hurdle) of this day maintenance mice.Inject for the second time afterwards mice to be killed in two days and be used for analyzing.All mices of having injected La serum are measured high level with the hIgG of mice La cross reaction by ELISA.Show and do not inject the same low of normal mouse serum (NMS) with the negative contrast of NHS in conjunction with background level (the 3rd hurdle).Use HEL (HEL) as negative control (the 4th hurdle) among the ELISA.Directly measure the serum levels of hIgG by quantitative ELISA, and find than the experiment of narration in the past much higher (Tran etc., 2002b).To conceived BALB/c mouse injection La serum and serum hIgG scope is 0.01 to 0.4g/L, the opsonic action (the 5th hurdle) of the fetus programmed cell death myocardial cell that it produces.Measure prostatic epithelium programmed cell death degree by the TUNEL algoscopy.The TUNEL that the digitized representation of indicating in the table detects in the obtainable prostatic epithelium layer in whole section ⁺Nuclear.Detected numerous TUNEL in the atrium to nearly all animal ⁺Particle is not counted.Compare with complete mice, the castrating mice has the tendency (the 6th hurdle) that increases programmed cell death.To castrating and detect combine (the 7th hurdle) of hIgG and programmed cell death prostate epithelial cell in three mices with the La serum injection one.In this case, detect numerous TUNEL of bright granule hIgG bag quilt at atrium ⁺Cell.Also see hIgG and TUNEL at epithelium and surface thereof ⁺The combination of cell.(adjoin) continuously the section immune labeled these immunocomplexs of prompting near have MHC-II ⁺And F4/80 ⁺Cell (not providing data).Significantly, animal or the injection La serum to injection NHS does not still have the animal of castrating not detect the combination of hIgG.As other contrast,, keep hIgG can detect serum levels to hIgG (prostate, spleen, liver, salivary gland) in sufficient background spaces of all zoometries of injecting and the blood vessel with La serum or NHS.As desired, measure numerous programmed cell death cell in spleen and the thymus, especially, spleen shows that tangible hIgG speckle dyeing (the blood tissues barrier is low in this tissue) does not have relevant TUNEL dyeing.In salivary gland, almost detect less than the programmed cell death cell after the castrating, with the programmed cell death that causes of castrating in the glandular epithelium be that the idea of prostate specific matches.

The tabulation of table 3. experimental result and narrative general introduction

Animal	Treatment	With bonded serum hIgG (the ELISA OD of recombinant antigen 405)		The serum levels of hIgG (g/L)	TUNEL (+) nuclear in the prostatic epithelium layer in each section	With the bonded hIgG of programmed cell death cell
							6HismLa	6HisHEL
		WT	Complete, La serum						1.850	0	3.2	2，3	-
WT	Complete, La serum			1.684	0	2.5	1	-
		WT	Castrating, La serum						1.534	0	1.8	＞30	+
WT	Castrating, La serum			1.685	0	2.7	8，10	-
		WT	Complete, NHS						0.007	0	1.5	N/A	-
WT	Castrating, NHS			0.014	0	0.9	1，3，10	-
		TRAMP	Castrating, La serum						1.588	0	2.4	＞30	-
TRAMP	Complete, La serum			1.389	0	1.6	5	-
		NMS	N/A						0.016	0	N/A	N/A	N/A

Embodiment 2

Anti--LA antibodies programmed cell death and non-viable non-apoptotic cell

External evoked programmed cell death and development, the omission that becomes gradually of programmed cell death cell, because lost the integrity of film, it shows cumulative respectively for nucleic acid and dna binding dye, the affinity of iodate third ingot (PI) and 7-amino-actinomycin D (7AAD).Compare with the PI combination, anti-La-antibody has the slower time with combining of programmed cell death.Yet the fluorescence intensity of observed anti-La-antibody staining shows that it combines with the programmed cell death cell with similar affinity, and no matter whether PI dyeing is height or moderate strength (Fig. 3).As follows, PI ^{Intermediate value}Subgroup comprises the programmed cell death body.Middle dyeing is not to end artifactitious result because no matter the change of PI concentration it all exist.

Therefore, to combine with the programmed cell death cell be the function that the film integrality as external programmed cell death process loses to anti--La antibody.Yet, anti--La antibody combines the passive binding events that is not simple mAb with the programmed cell death cell, because with the low log-fold of the painted fluorescence intensity ratio 3B9 of isotype contrast mAb Sal5 (anti--monoclonal antibody) fluorescence intensity, this show 3B9 combine with its target antigen people La/SS-B specificity (on the picture group and middle group, Fig. 4).Anti--tubulin mAb of recognizing cells support composition also proves and the bonded high level of programmed cell death cell, it than 3B9 in conjunction with less PI ^{Intermediate value}(picture is group down, Fig. 2) (total PI for the programmed cell death body ⁺46% of incident is with reference to 69%).

Illustrate some data shown in Fig. 4, and relatively do not have the percentage ratio of the cell of trypan blue at each time point, it is another indication (Fig. 5) of cell viability forfeiture.Undoubtedly, anti--La antibody is compared with the known mark thing and the cell viability forfeiture (trypan blue) of cell permeability (tubulin) with the bonded kinetics of programmed cell death cell, and this supports the film integrality forfeiture relevant with programmed cell death to allow the idea of anti--La antibodies.

Illustrating as Fig. 6, is that stable (picture is group down, Fig. 6), almost continues four days time, can (organize on the picture, Fig. 6) compare with the 3B9 staining power from the programmed cell death body size of programmed cell death Jurkat cell.

Using annexin V to survey the Phosphatidylserine that exposes on the ectoplast resists-La antibody and the bonded further dynamic analysis of programmed cell death Jurkat cell, the Phosphatidylserine that exposes on the ectoplast is the early sign of programmed cell death, and 7AAD is in conjunction with the DNA that omits cell, and it is a programmed cell death feature in late period.This is analyzed and to show once more that anti--La antibodies combine with 7AAD and be equal to, still than annexin V in conjunction with late generation (Fig. 7).This notion is showed in Fig. 8 in another way.When cell is that annexin V-positive and 7AAD-are when negative, anti--La antibodies is not obvious (R3 in the picture left hand group during the programmed cell death in early days, Fig. 8), it is permeable for 7AAD but in case cell becomes, then anti--La antibodies increase to high level (R1 in the picture left hand group, Fig. 8).

In order to emphasize to resist-the essential cell membrane integrity forfeiture of La antibodies, elementary non-viable non-apoptotic cell also confirms the eager combination (Fig. 9) for anti--La antibody.Note anti--La antibody staining and be exposed to not co (Fig. 9 A) of DNA on the non-viable non-apoptotic cell film (Fig. 9 B) or Phosphatidylserine.

Embodiment 3

Anti--La antibody is that caspase 3 is dependent with combining of programmed cell death body

Mention as embodiment 2, more detailed flow cytometry analysis indication resists-La antibodies programmed cell death body, it is characterized in that their less sizes and the inside complexity that reduces, because they contain the membrane-bound karyon composition and the organelle residue (Figure 10) of different proportion.In addition, anti--La antibodies is essential to prove that the programmed cell death bodily form is paired in.MCF-7 is the human breast cancerous cell line that does not have former caspase 3 genes.

Former caspase 3 is zymogen forms of conclusive executioner caspase 3, the cracking of a lot of functions and structural protein in its catalysis dead cell.The cracking of these proteinic caspase 3-mediations has contribution to the form outward appearance that the programmed cell death body forms, it with the programmed cell death cell division be several (or more) known be the less film bound fraction of programmed cell death body.Though the programmed cell death body formed during the MCF-7 cell can not prove cell death, by the gene transfection of former caspase 3 can be saved this phenotype in the MCF-7 cell.

As Figure 10 explanation,, produce the programmed cell death body and then in conjunction with resisting-La antibody with the gene transient transfection MCF-7 cell of former caspase 3.Therefore, resist-it is that caspase 3 is dependent that La antibody combines with the programmed cell death body.These programmed cell death bodies dye without 7AAD, and its preferentially dye DNA rather than RNA (picture lower-left hands group, Figure 10).In addition, when by the scattering standard evaluation, programmed cell death body size littler (picture bottom right hands group, Figure 10).

Further, research is with the MCF-7 of the stable gene transfection of former caspase 3.As shown in figure 11, make the MCF-7 cell programmed cell death that comprises control vector or former caspase 3 genes, and with 3B9, Alexa488 and iodate third ingot dyeing (Figure 11 A) of green fluorescence dye marker.Prove again to resist-La antibody combines with the programmed cell death body needs caspase 3 activity.The fluorescence microscope proof of these cells is expressed former caspase 3 rudiments of MCF-7 transfectant and is obviously separated 3B9 ⁺Programmed cell death body (Figure 11 C).On the other hand, the vehicle Control MCF-7 cell proof 3B9 less decentralized model (Figure 11 C) that dyes, this and observed 3B9 dyeing match (the upper right hands group of picture, Figure 11 A) that extensively distribute to the flow cytometer of vehicle Control MCF-7 cell.On the contrary, as in Figure 10, caspase 3 activity are brought the painted restricted mode of 3B9 (picture bottom right hands group, Figure 11 A), and these prompting caspase 3 activity cause the antigenic even separation of La/SS-B in the programmed cell death body.

As shown in figure 12, utilize the same focal argon laser scanning microscope proof of programmed cell death Jurkat cell anti--the La antibody staining neither with the programmed cell death cell membrane dyeing that transforms Phosphatidylserine overlapping (detecting) according to annexin V, also not to DNA dyeing (detecting) (Figure 13 A) according to TOPRO3.In addition, a succession of terrace cut slice confirms that anti--La antibody staining takes place the cytoplasmic region that runs through dead cell.With 3B9 dyeing is specific, because use isotype tester mAb, Sal5 does not almost find any dyeing (Figure 13 B).PARP, its be the karyon antigen (comprising about 2% karyon protein) that enriches and during programmed cell death caspase 3 when detecting, adopt dyeing pattern (Figure 12 C) also with its cracking similar in appearance to 3B9 with specificity mAb.The kytoplasm (Figure 13) of these data are pointed out together anti--La antibody ' loading ' dead cell.

To karyon antigen specific other mAb of fodrin for example, therefore it be formed with contribution to the programmed cell death body also by caspase 3 cracking, uses the similar dyeing pattern of showing with 3B9 to programmed cell death Jurkat cell dyeing (Figure 14).For PCNA and the specific mAb of lamin B are also observed similar dyeing pattern.These mAb identity basis 7AAD detect not with the DNA co.In fact, 7AAD tends to be limited to periphery programmed cell death bleb.On the contrary, be to a certain extent with the localized evidence that runs through the programmed cell death cell of 7AAD dyeing with anti--beta tubulin mAb dyeing.

Anti--La antibody also in conjunction with the elementary T cell of programmed cell death, comprises most of peripheral blood lymphocytes (PMBC).Yet, with elementary T cell bonded anti--fluorescence intensity of La antibody compares little only about half of to several times (one half-logfold) that pernicious Jurkat T cell observation arrives, even using T cell mitogen in advance, conconavalin A activates PBMC (Figure 15).

Embodiment 4

Other monoclonal antibodies of anti-other karyons and ribose nuclear antigen are also in conjunction with the programmed cell death cell

According to observed in the research of focal argon laser scanning microscope, flow cytometry proves that multiple mAb is in conjunction with similar kinetics and pattern.Anti--tubulin mAb is as the bonded tester of kytoplasm (Figure 16) in the permeable programmed cell death cell.Similarly, some specificitys among these mAb are in conjunction with the programmed cell death body (Figure 17) with formation after the former caspase 3 gene transfection MCF-7 cells.

Embodiment 5

Other antibody of anti-people LA/SS-B also detect the programmed cell death cell

Studied the people anti--La autoantibody (Figure 18) and people La/SS-B had specific another kind of mAb, clone SW3 is to the combination (Figure 19) of programmed cell death cell.

Embodiment 6

Anti--LA antibodies is from the elementary and pernicious programmed cell death cell of people and rodent species

Except in conjunction with the programmed cell death primary human cell (Figure 15), anti--La antibody is also in conjunction with wherein with the programmed cell death primary cell of dexamethasone or dead mice of the external evoked programmed cell death of staurosporine and rat chest gland.Anti--La antibody respond the inductive programmed cell death of any stimulus object and specificity in conjunction with PI ⁺Thymocyte cell (Figure 20).Use the mAb of antiproliferative cell karyon antigen (PCNA) to observe similar binding pattern.Anti--La antibody is also in conjunction with the programmed cell death tumor cell (Figure 21) from the rodent species, the tumor cell that comprises experience programmed cell death in the response cytotoxic drug body, and multiple programmed cell death people and monkey tumor cell line (Figure 22).

Those skilled in the art understand that the present invention described herein also changes easily and modifies except specifically described.Understand and the present invention includes all such change and modifications.The present invention comprise also that this description individually or is briefly pointed out or indicated institute in steps with feature, compositions and chemical compound, and two or several any He all combinations of described step or feature.

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Claims (55)

1. to the experimenter or from the method for described experimenter's biology sample detection programmed cell death cell, described method comprises uses cell or cell extract and the screening interacting molecule-karyon molecular complex formation that contacts described experimenter or described biological sample at the interacting molecule of karyon molecule or its antigen part, and the non-karyon location of wherein said complex is the indication of programmed cell death cell.

2. the analytical method of programmed cell death cell in the detection of biological sample, described method comprises following step:

(1) interacting molecule at karyon molecule or its antigenic determinant is contacted with suspecting the biological sample that contains the cell of expressing described karyon molecule; With

(2) complex that forms in the step (1) is carried out signal detection step,

Wherein detecting non-karyon interacting molecule-karyon molecular complex formation is the indication of programmed cell death cell.

3. the experimenter is detected the method for programmed cell death cell, described method comprises to described patient introduces the interacting molecule at karyon molecule or its antigenic determinant of using the reporter molecule labelling, make the molecule of labelling select the interacting molecule of part to propagate at blood circulation, then described patient is carried out the reporter molecule detection means and detect to determine the interacting molecule position by blood circulation or to described patient's input.

4. according to each method of claim 1-3, wherein said karyon molecule is Ro52, Ro60, La/SS-B, gelsolin, α-fodrin, fibrillarin, U1 micronuclear ribonucleoprotein matter (U1 snRNP), heteronuclear ribonucleoprotein matter (hnRNP), lamin B, poly-(ADP-ribose) polymerase (PARP), proliferating cell nuclear antigen (PCNA), SC-35 splicing factor, Smith (Sm) antigen.

5. according to the method for claim 4, wherein said karyon molecule is La.

6. according to each method of claim 1-5, wherein said non-karyon location is kytoplasm or relevant with the programmed cell death body.

7. according to claim 4 or 5 or 6 each methods, wherein said interacting molecule is an immunointeractive molecule.

8. according to the method for claim 7, wherein said immunointeractive molecule is an antibody.

9. method according to Claim 8, wherein said antibody is monoclonal antibody.

10. according to each method of claim 1-9, wherein said programmed cell death cell is programmed cell death heart cell, neurocyte or lymphoid cell.

11. according to each method of claim 1-9, wherein said programmed cell death cell is a tumor cell.

12. according to the method for claim 11, wherein said tumor cell is central nervous system's tumor, retinoblastoma, neuroblastoma and other department of pediatrics tumors, head and neck cancer, breast and carcinoma of prostate, pulmonary carcinoma, renal carcinoma, esophagus gastric cancer, hepatocarcinoma, the ductus pancreaticus biloma forms, colorectal carcinoma, cervical cancer and anus cancer, uterus and other reproductive tract cancers, carcinoma of urethra, the blastocyte tumor, ovarian cancer, unknown primary carcinoma, the human immune deficiency associated malignancies, lymphoma, leukemia, malignant melanoma, sarcoma, endocrine tumor, mesothelioma and other pleural neoplasms, neuroendocrine tumor and cancer sample tumor.

13. method according to claim 12, wherein said head and neck cancer is a squamous cell carcinoma, described pulmonary carcinoma is small cell lung cancer and nonsmall-cell lung cancer, described renal carcinoma is a renal cell adenocarcinoma, described pancreas tumor is the adenocarcinoma islet cell tumor, and described blastocyte tumor is carcinoma of testis or ovarian cancer, and described ovarian cancer is an epithelial ovarian cancer, and described human immune deficiency associated malignancies is the Kaposis sarcoma, and described endocrine tumor is a thyroid tumor.

14. the experimenter is diagnosed or monitors with unusual method that do not expect or that other unsuitable cell programmed cell death is the disease of feature, described method comprises the karyon molecule used at described karyon molecule or its antigenic determinant or epi-position-contact described experimenter or from the cell or the cell extract of described experimenter's biological sample in conjunction with the interacting molecule of effective dose, and qualitatively or quantitatively determine karyon molecule-immunointeractive molecule complex and form, the non-karyon location of wherein said complex is the indication of cell programmed cell death.

15. according to the method for claim 14, wherein said karyon molecule is Ro52, Ro60, La/SS-B, gelsolin, α-fodrin, fibrillarin, U1 micronuclear ribonucleoprotein matter (U1 snRNP), heteronuclear ribonucleoprotein matter (hnRNP), lamin B, poly-(ADP-ribose) polymerase (PARP), proliferating cell nuclear antigen (PCNA), SC-35 splicing factor, Smith (Sm) antigen.

16. according to the method for claim 15, wherein said karyon molecule is La.

17. according to each method of claim 14-16, wherein said non-karyon location is kytoplasm or relevant with the programmed cell death body.

18. according to each method of claim 15-17, wherein said interacting molecule is an immunointeractive molecule.

19. according to the method for claim 18, wherein said immunointeractive molecule is an antibody.

20. according to the method for claim 19, wherein said antibody is monoclonal antibody.

21. according to each method of claim 14-20, wherein said programmed cell death cell is programmed cell death heart cell, neurocyte or lymphoid cell.

22. according to each method of claim 14-20, wherein said disease is cardiac muscle or cerebral tissue infraction, autoimmune and other inflammation diseases, virus disease, AIDS for example, neurodegenerative disease, for example presenile dementia or parkinson disease, acute solid organ or bone marrow transplantation rejection, tissue injury that chemotherapy or radiotherapy cause (' mucositis ') or tumor resemble tumor.

23. according to the method for claim 22, wherein said disease is central nervous system's tumor, retinoblastoma, neuroblastoma or other department of pediatrics tumors, head and neck cancer, breast and carcinoma of prostate, pulmonary carcinoma, renal carcinoma, esophagus gastric cancer, hepatocarcinoma, the ductus pancreaticus biloma forms, colorectal carcinoma, cervical cancer and anus cancer, uterus and other reproductive tract cancers, carcinoma of urethra, the blastocyte tumor, ovarian cancer, unknown primary carcinoma, the human immune deficiency associated malignancies, lymphoma, leukemia, malignant melanoma, sarcoma, endocrine tumor, mesothelioma and other pleural neoplasms, neuroendocrine tumor and cancer sample tumor.

24. method according to claim 23, wherein said head and neck cancer is a squamous cell carcinoma, described pulmonary carcinoma is small cell lung cancer or nonsmall-cell lung cancer, described renal carcinoma is a renal cell adenocarcinoma, described pancreas tumor is the adenocarcinoma islet cell tumor, and described blastocyte tumor is carcinoma of testis or ovarian cancer, and described ovarian cancer is an epithelial ovarian cancer, described human immune deficiency associated malignancies is the Kaposis sarcoma, and described endocrine tumor is a thyroid tumor.

25. according to each method of claim 1-24, wherein said experimenter is a mammal.

26. according to the method for claim 25, wherein said mammal is the people.

27. at the interacting molecule of karyon molecule be used to prepare detection from patient's biological sample programmed cell death cell quantitatively, the purposes of sxemiquantitative or qualitative diagnostic reagent kit.

28. according to the purposes of claim 27, wherein said karyon molecule is Ro52, Ro60, La/SS-B, gelsolin, α-fodrin, fibrillarin, U1 micronuclear ribonucleoprotein matter (U1 snRNP), heteronuclear ribonucleoprotein matter (hnRNP), lamin B, poly-(ADP-ribose) polymerase (PARP), proliferating cell nuclear antigen (PCNA), SC-35 splicing factor, Smith (Sm) antigen.

29. according to the purposes of claim 27, wherein said karyon molecule is La.

30. according to the purposes of claim 28 or 29, wherein said interacting molecule is an antibody.

31. according to the purposes of claim 30, wherein said antibody is monoclonal antibody.

32. to experimenter's therapeutic and/or prophylactic treatment is the method for the disease of feature with the cell programmed cell death, described method is included in to be enough to treat under the time and condition of described disease, described experimenter is used the interacting molecule at karyon molecule or its antigen part of effective dose, and described interacting molecule is connected with therapeutic or preventative effector structure, bonding or association.

33. according to the method for claim 32, wherein said karyon molecule is Ro52, Ro60, La/SS-B, gelsolin, α-fodrin, fibrillarin, U1 micronuclear ribonucleoprotein matter (U1 snRNP), heteronuclear ribonucleoprotein matter (hnRNP), lamin B, poly-(ADP-ribose) polymerase (PARP), proliferating cell nuclear antigen (PCNA), SC-35 splicing factor, Smith (Sm) antigen.

34. according to the method for claim 33, wherein said karyon molecule is La.

35. according to the method for claim 33 or 34, wherein said interacting molecule is an immunointeractive molecule.

36. according to the method for claim 35, wherein said immunointeractive molecule is an antibody.

37. according to the method for claim 36, wherein said antibody is monoclonal antibody.

38. according to each method of claim 32-37, wherein said programmed cell death cell is programmed cell death heart cell, neurocyte or lymphoid cell.

39. according to each method of claim 32-37, wherein said disease is cardiac muscle or cerebral tissue infraction, autoimmune and other inflammation diseases, virus disease, AIDS for example, neurodegenerative disease, for example presenile dementia or parkinson disease, acute solid organ or bone marrow transplantation rejection, tissue injury that chemotherapy or radiotherapy cause (' mucositis ') or tumor resemble tumor.

40. according to the method for claim 39, wherein said disease is central nervous system's tumor, retinoblastoma, neuroblastoma or other department of pediatrics tumors, head and neck cancer, breast and carcinoma of prostate, pulmonary carcinoma, renal carcinoma, esophagus gastric cancer, hepatocarcinoma, the ductus pancreaticus biloma forms, colorectal carcinoma, cervical cancer and anus cancer, uterus and other reproductive tract cancers, carcinoma of urethra, the blastocyte tumor, ovarian cancer, unknown primary carcinoma, the human immune deficiency associated malignancies, lymphoma, leukemia, malignant melanoma, sarcoma, endocrine tumor, mesothelioma and other pleural neoplasms, neuroendocrine tumor or cancer sample tumor, and described treatment is the growth of decrement regulate tumor cell.

41. method according to claim 40, wherein said head and neck cancer is a squamous cell carcinoma, described pulmonary carcinoma is small cell lung cancer or nonsmall-cell lung cancer, described renal carcinoma is a renal cell adenocarcinoma, described pancreas tumor is the adenocarcinoma islet cell tumor, and described blastocyte tumor is carcinoma of testis or ovarian cancer, and described ovarian cancer is an epithelial ovarian cancer, described human immune deficiency associated malignancies is the Kaposis sarcoma, and described endocrine tumor is a thyroid tumor.

42. according to claim 40 or 41 each methods, wherein said tumor is a metastatic carcinoma, and described treatment is the growth of decrement regulate tumor cell.

43. according to each method of claim 32-42, wherein said experimenter is a mammal.

44. according to the method for claim 43, wherein said mammal is the people.

45. according to each method of claim 32-44, wherein said effector structure is selected from:

-cytokine

-chemotactic factor

-macrophage, dendritic cell or t cell activation agent; Perhaps

-toxin.

46. according to the method for claim 45, wherein said macrophage activation agent is N-formoxyl-methionyl-leucyl-phenylalanine or antibacterial lipopeptid.

47. according to the method for claim 46, wherein said antibacterial lipopeptid is JBT2002 or its derivant or analog.

48. according to the method for claim 45, wherein said toxin is a radiosiotope.

49. according to the method for claim 48, wherein said radiosiotope is the alpha particle emitter, beta particle emitter or gamma particles emitter.

50. according to the method for claim 49, wherein said alpha particle emitter is Tb-149 or Bi-213.

51. according to the method for claim 45, wherein said toxin is a Ricin, prodrug or biopharmaceuticals.

52. according to the method for claim 51, wherein said prodrug is the prodrug invertase of antibody-orientation.

53. according to the method for claim 51, wherein said biopharmaceuticals is a catalytic antibody.

54. be used to prepare the purposes of the medicine of treatment experimenter disease with the link coupled karyon interaction of molecules of effector structure molecule, described disease is a feature with the cell programmed cell death, wherein said effector structure is treated described disease.

55. contain the pharmaceutical composition of regulator and one or more pharmaceutical acceptable carriers and/or diluent, described regulator is called active component.

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