CN1678347A

CN1678347A - Antibodies and uses thereof

Info

Publication number: CN1678347A
Application number: CNA038204029A
Authority: CN
Inventors: A·莱瓦农; R·本-莱维; D·普拉克辛; E·桑顿; Y·哈加尔; H·霍奇马－哈伊姆
Original assignee: Savient Pharmaceuticals Inc
Current assignee: Savient Pharmaceuticals Inc
Priority date: 2002-07-01
Filing date: 2003-06-30
Publication date: 2005-10-05
Also published as: EP1534332A2; KR20060011925A; PL374439A1; BR0312483A; IL166063A0; WO2004003166A3; AU2003263764A1; EP1534332A4; CA2491363A1; JP2005536199A; MXPA05000272A; WO2004003166A2; RU2005101621A

Abstract

The present invention provides antibodies or fragments thereof that bind to cancer cells and are important in physiological phenomena, such as cell rolling and metastasis. Therapeutic and diagnostic, prognostic or staging methods and compositions using such antibodies or fragments thereof are also provided. The methods and compositions according to the present invention can be used in diagnosis of and therapy for such diseases as cancer, including tumor growth and metastasis, leukemia, autoimmune disease, and inflammatory disease.

Description

Antibody and application thereof

Invention field

The present invention relates to and the bonded antibody of special epi-position, described epi-position is present on the cell, as cancerous cell, transitional cell, leukaemia, leukocyte and platelet, in physiology's phenomenons such as cell rolling, transfer, inflammation and autoimmune disease, play an important role.Specifically, described antibody can have active anticancer, antimetastatic activity, anti-leukocythemia liveness, antiviral activity, anti-infection activity and/or anti-other disease activity, the disease that causes as the protein-protein interaction of diseases associated with inflammation, autoimmune disease, cardiovascular disease (as myocardial infarction, retinal diseases) and dependence sulphation tyrosine.

Background of invention

Antibody, phage display and tissue target to

The tissue selectivity target therapeutic agent is a direction emerging in the pharmaceutical industries.Now designed new cancer treatment method, when increasing treatment specificity and effectiveness, reduced toxicity, thereby promoted overall effectiveness based on targeting.Attempted being used for toxin, radioactive nucleus thuja acid and chemotherapy conjugate target tumor at the mouse monoclonal antibody (MAb) of tumor associated antigen.In addition, differentiation antigen such as CD19, CD20, CD22 and CD25 have been developed the cancer specific target as treatment hemopoietic system malignant tumor.

Though carried out detailed research, this method has some limitation.A defective is ten fens difficulties of MAb of separating suitable demonstration selective binding.Second defective is the prerequisite as successful separation antibody, needs high antibody mediated immunity originality thing.The 3rd defective is that end product has the non-human sequence, and this can cause immunne response; As when giving human mice MAb, can produce human anti-mouse antibody (HAMA) and reply.HAMA replys and usually causes plasma half-life to shorten, and can not treat repeatedly, has therefore reduced the therapeutic value of this antibody.The defective in back has evoked the interest of the chimeric or Humanized monoclonal antibodies in engineered production mice source, and the interest of exploit person antibody.It is the monospecific antibody type that can only separate anti-known purifying antigen that this method also has a defective.And this method selectivity is not high enough, and its isolated antibody is at the cell surface marker that is present on normal cell and the malignant cell.

There are many factors can influence MAb treatment treatment for cancer effect.These factors comprise the accessibility of specificity and level, antigenic heterogeneity and the tumor enclosed mass of antigen presentation on the tumor cell.The reactivity of leukemia and the treatment of lymphoma antagonist is better than entity tumor (for example cancer).MAb leukaemia and lymphoma cell rapid and in the blood flow combine, and enter the malignant cell in the lymphoid tissue easily, and therefore the treatment based on MAb has good result to lymph tumor.Idealized system need identify that identification produces the stem cell surface labelling of pernicious daughter cell.

Phage library has been used for screening and the bonded strand at random of target protein variant fragment (scFv), and described target protein is the isolating albumen that is predetermined, as antibody, hormone and receptor.The application in antibody display libraries, especially phage scFv library promotes the another kind of method of finding the unique molecular of targeting special cells surface portion, even this cell surface part is not understood by people yet or character is not bright.

Leukemia, lymphoma and myeloma relate to the uncontrolled growth of cell for rising in bone marrow and adenoid cancer.Acute lymphoblastic leukemia (ALL) is a different substantiality disease, by specific clinical and immunological characteristic definition.Similar to the ALL of other form, the definite cause of disease of most of B cell ALL (B-ALL) is unknown; But this disease is changed by the homotropic inheritance in a kind of cell DNA and causes that this change causes unusual and persistence propagation under many situations.Compare with other leukaemic, B-ALL patient's prognosis is significantly relatively poor, is not always the case in child and adult.B cell CLL (B-CLL) is an example of chronic lymphocytic leukemia (CLL), is a kind of leukemia of slower development, and it is characterized by lymphocyte quantity increases.Acute myeloid leukemia (AML) is a kind of heterogeneous tumor, comprises CFU-GM, and CFU-GM under normal circumstances finally can form medullary system noble cells (erythrocyte, granulocyte, mononuclear cell and platelet).The same with other form tumor, AML relates to homotropic inheritance and changes, and this change causes undifferentiated relatively blastocyte to replace the medullary cell of normal differentiation, shows as to have a kind or multiple early stage medullary system differentiation type.AML originates from bone marrow usually, the secondary hemopoietic organ of shorter mention.AML mainly influences the adult, and the sickness rate peak is at 15-40 between year, but known it also influence child and older adult.After making a definite diagnosis, nearly all AML patient needs to treat immediately to obtain clinical remission, even there is not circulation not break up the patient of the unusual sign of blastocyte.

So far developed the active antitumor cell MAb of various lysises.The extracellular domain of mice MAbmuMab4D5 antagonism HER2 (P185), the propagation that can significantly suppress the human tumor cells of overexpression HER2, it by humanization to produce medicine HERCEPTIN  (trastuzumab), this medicine is used for the treatment of human breast carcinoma (U.S. Patent number 5 through the FDA permission, 821,337 and 5,720,954).In conjunction with after, antibody can suppress to rely on the growth of tumour cell of HER2 growth factor receptors.In addition, CD 20 antagonizing Chimeric antibody has also been passed through FDA permission (U.S. Patent number 5,843,439) recently, and this antibody causes periphery B cell to exhaust rapidly, comprises lymphoma B cell.This antibody combines the cracking that causes relying on complement with target cell.This product is recently by permission, currently is used for the treatment of low B cell Fei Huojiejin lymphomas clinically.

Some other humanization and chimeric antibody developing or clinical trial in.For example, combine (Sievers et al. with the Humanized immunoglobulin (Ig) of CD33 antigenic specificity reaction with anticarcinogen calicheamicin CMA-676, Blood Supp.308:504a (1997)), CD33 be expressed on the normal marrow cell and the bone marrow leukemia cells of most of kinds on.This conjugate is called medicine MYLOTARG  again, has obtained FDA permission (Caron et al., Cancer Supp.73:1049-56 (1994)) recently.According to its lysis activity, another anti-CD 33 antibody (HumM195) that is in clinical trial combines with some cytotoxic drug, comprise gelonin toxin (McGraw et al., Cancer Immunol.Immunother.39:367-74 (1994)) and radiosiotope ¹³¹I (Caron et al., Blood 83:1760-68 (1994)), ⁹⁰Y (Jurcic et al., Blood Supp.92:613a (1998)) and ²¹³Bi (Humm etal., Blood Supplement 38:231P (1997)).The chimeric antibody of anti-human leucocyte antigen CD45 (cHuLym3) also is in clinical trial, is used for the treatment of human leukemia and lymphoma (Sun etal., Cancer Immunol.Immunother 48:595-602 (2000)).In external test, ADCC (antibody dependent cellular mediated cell toxicity) can be observed specific cell cracking (Henkart, Immunity 1:343-46 (1994) in measuring; Squier and Cohen, Current Opin.Immunol.6:447-52 (1994)).

These therapeutic antibodies make it have higher affinity to target through special engineered, and are more stable, and have best bio distribution.Referring to as Presta, Current Pharma.Biotechnol., 3:237-56 (2002); Presta et al., Biochem.SocietyTransactions, 30 (4): 487-90 (2002).

Compare with the structure of mouse monoclonal humanization and chimeric antibody, the application of display technique of bacteriophage allows the separation of complete human sequence scFv.Developed the fully human antibodies based on scFv clone's anti-people TGF beta 2 receptor recently, described clone promptly derives from display technique of bacteriophage.This scFv can be converted into the whole person IgG4 (Thompson etal., J.Immunol.Meth.227:17-29 (1999)) that combines competition with TGF β 2, has very strong antiproliferative activity.Display technique of bacteriophage is as technology well known by persons skilled in the art, in following publication by more detailed description: Smith, Science 228:1315 (1985); Scott et al., Science249:386-90 (1990); Cwirla et al., PNAS 87:6378-82 (1990); Devlin etal., Science 249:404-06 (1990); Griffiths et al., EMBO is (14) J.13: 3245-60 (1994); Bass et al., Proteins 8:309-14 (1990); McCafferty etal., Nature 348:552-54 (1990); Nissim et al., EMBO is (1994) J.13:692-98; U.S. Patent number 5,427,908,5,432,018,5,223,409 and 5,403,484, lib.

The part of isolating scFv antibody molecule

Platelet, Fibrinogen, GPIb, selection albumen and PSGL-1 (P-selects protein sugar protein ligands-1) play an important role in some pathogenic state or disease stage; as unusual or pathogenic inflammation, unusual or pathogenic immunoreation, autoimmune response, transfer, unusual or pathogenic adhesion, thrombosis and/or restenosis, and the unusual or gathering of causing a disease.Therefore, combine with platelet and above-mentioned molecule or the antibody of cross reaction can be used for diagnosing and treating disease and the imbalance that relates to these and other pathogenic disease states.

Platelet

Platelet is a blood system typical characteristic composition, plays some important function in hemostasis, thrombosis and/or restenosis.The blood vessel injury startup is known as the hemostatic process, and described process is a feature with a series of successive incidents.Initial reaction to damaged blood vessels is the involved area that platelet is attached to vascular inner surface.Next step be the multilamellar platelet aggregation on the adherent platelet in front, form tampon, occluding vascular.Tampon is further strengthened by the deposition of fibrin polymerization body.Coagulate and determine or bolt just degraded after injury repairing.

The circulation platelet is the cytoplasmic granule that discharges from the periphery megalokaryocyte.Platelet plays an important role in hemostasis.When blood vessel injury, platelet adhesion is to the damaged tissues surface, and (adhesion) is bonded to each other.This chain of events takes place rapidly, forms structureless (being commonly referred to platelet bolt or thrombosis) at vascular injury site.Adhesion is also referred to as gathering, can by various materials or agonist is external causes, as collagen, adenosine diphosphate (ADP) (ADP), epinephrine, serotonin and ristocetin.Gathering is for measuring a kind of in numerous in vitro testses that platelet function carries out.

The importance of platelet in transfer

Neoplasm metastasis perhaps is the greatest factor of restriction cancer patient existence.The data of accumulation point out that tumor cell and the interactional ability of host's platelet have been represented one of the necessary determiner of transfer (Oleksowicz, Thrombosis Res.79:261-74 (1995)).When the metastatic carcinoma cell enters blood flow, form the many cells complex that surrounds tumor cell by platelet and leukocyte.This complex can be described as microemboli, helps tumor cell to hide immune system.The platelet bag is needed platelet to express P selection albumen by tumor cell.

Verified, the ability of tumor cell and platelet aggregation is relevant with the metastatic potential of tumor cell, in the rodent model tumor is caused that the inhibition of platelet aggregation is relevant with the inhibition of transfer.Verified, tumor cell and hematoblastic interaction relate to film adhesion molecule and agonist secretion.Identify tumor cell and fastened the expression of the relevant platelet glycoprotein of immunity.Verified, platelet immunity associated glycoprotein GPIb, GPIIb/IIIa, GPIb/IX and beta 2 integrin alpha _VSubunit expression in the breast tumor cell line surface (Oleksowicz, (1995), the same; Kamiyama et al., J.Lab.Clin.Med.117 (3): 209-17 (1991)).

Gasic et al. (PNAS 61:46-52 (1968)) shows, the thrombocytopenia that antibody causes significantly reduces quantity and volume (Karpatkin et al., J.Clin.Invest.81 (4): the 1012-19 (1988) of the metastasis of CT26 adenocarcinoma of colon, Lewis lung cancer and the generation of B16 melanoma; Clezardin et al., Cancer Res.53 (19): 4695-700 (1993)).In addition, express single chain polypeptide (60kd) on the skin covering of the surface of discovery hel cell, described polypeptide chain and GPIb are closely related, are equivalent to the glycosylated GPIb α of imperfect or unusual O subunit (Kieffer et al., J.Biol.Chem.261 (34): 15854-62 (1986)).

The GPIb complex

Each step in the hemostasis all needs the existence of receptor on the platelet surface.A kind of important receptor is a glycoprotein I B-IX complex (being also referred to as CD42) in the hemostasis.This receptor mediates the damaged part of platelet adhesion (initial adhering to) to blood vessel wall by in conjunction with the subendothelium von Willebrand factor (vWF).It also plays a crucial role in 2 important platelet functions in hemostasis in addition: (a) the inductive platelet aggregation of high shear force in the stricture of artery zone; (b) platelet activation of low concentration thrombin induction.

The GPIb-IX complex is a kind of main component of platelet plasma membrane outer surface.This complex comprises 3 and strides GPIb 130kDa α chain and the 25kDa β chain that membrane polypeptides-disulfide bond connects, and the GPIX of non-covalent connection (22kDa).For effectively cell surface expression and CD42 complex function, all subunit equimolar amountss are present on the platelet membrane, are illustrated in that to express fully on the plasma membrane be that the correct assembling of 3 subunits is necessary.The α chain of GPIb is made up of 3 different structure territories: (1) comprises rich leucine repetitive sequence and the cysteine spherical N end peptide territory in conjunction with flanking sequence; (2) the huge glycopeptide of the mucin sample territory of high glycosylation; (3) comprise GPIb α disulfide bond and to stride film relevant with the film of kytoplasm sequence C end territory.

Some evidence shows that the vWF of GPIb-IX complex and thrombin are positioned at GPIb α N end in conjunction with the territory and comprise about 300 amino acid whose spherical region.Because human blood platelets GPIb-IX complex is mediation platelet function and reactive crucial membrane receptor, make platelet adhesion in damaged blood vessels in conjunction with vWF by subcutaneous in the GPIb identification.In addition, vWF combines with GPIb α and also causes platelet activation, and this may relate to the interaction of cytoplasm domain and cytoskeleton or the phospholipase A2 of GPIb-IX.In addition, GPIb α comprises α thrombin high affinity binding site, and this site promotes platelet activation by unknown mechanism.

The N of GPIb α holds spherical territory to comprise negative charge aminoacid bunch.Some evidence shows that in GPIb-IX and platelet GPIb α that transfection CHO cell is expressed, 3 tyrosine residues that comprise in this territory are by sulphation (Tyr-276, Tyr-278 and Tyr-279).

The albumen sulphation

The albumen sulphation is common post translational modification, and it relates to the covalently bound of enzyme catalysis sulphuric acid and sugared side chain or polypeptide backbone.This modification can be crossed over Gorky's chamber.Sulphation albumen comprises secretory protein, the particulate albumen of targeting and plasmalemma protein extracellular region.Tyrosine be a kind of known can Sulfated amino acid residue (Kehoe et al., Chem.Biol.7:R57-61 (2000)).Other aminoacid such as threonine also can be by sulphations, especially in the disease cell.

Found some proteic tyrosine by sulphation, but the situation that has 3 or more poly-sulfated tyrosine like that on a polypeptide that the GPIb that coexists go up to find is uncommon.GPIb α (CD42) is expressed by platelet and megalokaryocyte, and by adhering to and roll at subendothelium in conjunction with vWF mediation platelet, its N end regions also comprises numerous negative charges.This peracidity and hydrophilic environment are considered to Sulfated prerequisite, near because the tyrosine (Bundgaard etal., J.Biol.Chem.272:21700-05 (1997)) tyrosyl albumen sulfotransferase specific recognition and the sulphation acidic amino acid residue.The complete sulphation in GPIb alpha acid district produces in the zone-19 amino acid whose length that has remarkable negative charge density with 13 negative charges-become candidate locus with other albumen electrostatic interaction.

Sulphation N end tyrosine also is considered to influence the effect of CC-chemokine receptor such as CCR5, described receptor relates to human and simian immunodeficiency virus (HIV-1, HIV-2 and SIV) target approach cell as the coreceptor of relevant 7 transmembrane segments (7TMS) receptor.For example, sulphation N end tyrosine is believed to be helpful in combining of CCR5 and MIP-1 α, MIP-1 β and HIV-1 gp120/CD4 complex, helps HIV-1 to enter the cell of expression CCR5 and CD4.But another important HIV-1 coreceptor CXCR4 also sulphation (Farzan et al., Cell 96 (5): 667-76 (1999)).Compare with relying on entering of CCR5, in the HIV-1 that relies on CXCR4 entered, the Sulfated effect of tyrosine was more not remarkable; Therefore shown the possible effect in CXC chemotactic factor family of tyrosine sulphation, and HIV-1 utilizes the general difference (Farzan et al., J Biol.Chem.277 (33): 29,484-89 (2002)) of CCR5 and CXCR4.

Select albumen and PSGL-1

It is the member of adhesion molecule family that P, E and L select albumen, and one of function of described molecule family is the rolling of mediated leucocytes on blood vessel endothelium.P selects albumen to preserve in platelet with particle form, is transported to the surface after platelet is by thrombin, histamine, phorbol ester or the activation of other stimulation molecule.P selects albumen also to be expressed on the activation endotheliocyte.E selects protein expression on endotheliocyte, and L selects protein expression on neutrophil(e) cell, mononuclear cell, T cell and B cell.

PSGL-1 (being also referred to as CD162) selects albumen and L to select proteic mucin glycoprotein ligand for P selects albumen, E, and it and GPIb have analog structure (Afshar-Kharghan etal. (2001), the same).PSGL-1 is the homodimer that cystine linkage connects, and has PACE (pairing basic amino acid invertase) restriction enzyme site.Before ten aggressiveness of 10-16 proline rich, serine and threonine, PSGL-1 also has 3 potential tyrosine sulphation sites.Born of the same parents' outer part of PSGL-1 contains 3 N linked glycosylation sites and numerous sialylated and fucosylated O joins oligosaccharide branch (Moore et al., J.Biol.Chem.118:445-56 (1992)).Most of N polysaccharide sites and many O polysaccharide site are occupied.Measured the O glycan structures of the PSGL-1 that derives from people HL-60 cell.The hypotype of these O polysaccharide is core-2, and its sialylated and fucosylated structure is and selects protein binding necessary.The tyrosine sulphation in PSGL-1 aminoterminal zone also is to select albumen and L to select protein binding necessary with P.In addition, can translate the N end propetide of back excision in addition.

PSGL-1 in the HL60 cell has 361 residues, 267 extracellular region residues are wherein arranged, stride film residue and 69 intracellular region residues for 25, form homodimer or the heterodimer (Afshar-Kbarghan et al., Blood 97:3306-12 (2001)) that disulfide bond connects at cell surface.The sequence of coding PSGL-1 is arranged in single exon, therefore alternative splicing can not be arranged.But, PSGL-1 in HL60 cell and the most cells system has 15 continuous multiple 10 residue consensus sequences at its extracellular region, and polymorphonuclear leukocyte, mononuclear cell and some other cell line have 14 and 16 multiple these sequences in (comprising most of natural leukocyte).

The last PSGL-1 of neutrophil(e) cell is expressed as dimer, and apparent molecular weight is 250kDa and 160kDa, and dimeric apparent molecular weight is about 220kDa on HL60.When analyzing under reducing condition, each subunit is by incomplete reduction (reduced by half).Its molecular weight difference is because due to the molecule polymorphism, described polymorphism is that ten aggressiveness number of iterations differences cause (Leukocyte Typing VI.Edited by T.Kishimoto et al. (1997)).

Most of blood leucocytes are as neutrophil(e) cell, mononuclear cell, leukocyte, B cell subsets and all T cellular expression PSGL-1 (Kishimoto et al. (1997), the same).The rolling of PSGL-1 mediated leucocytes on activation endothelium, activated blood platelet, other leukocyte and on the inflammation part also mediates the rolling of neutrophil(e) cell on P selection albumen.PSGL-1 also can interact by select protein mediated neutrophil(e) cell-neutrophil(e) cell in conjunction with L, thus transmitting inflammation (Snapp et al., Blood 91 (1): 154-64 (1998)).

Leukocyte rolls very important in inflammation, P selects albumen (to be expressed on activation endothelium and the platelet, can be fixed on damage location) and PSGL-1 between interaction leukocyte is adhered on blood vessel wall and roll (Ramachandran et al., PNAS 98 (18): 10166-71 (2001); Afshar-Kharghan et al. (2001), the same).Cell rolling is also very important in transfer, and P on the endotheliocyte and E select albumen to be considered to and can to combine with transitional cell, therefore promote it to be seeped into the surrounding tissue outside blood flow.

Therefore, find that PSGL-1 is present on all leukocyte: neutrophil(e) cell, mononuclear cell, lymphocyte, activation periphery T cell, granulocyte, oxyphil cell, platelet, the positive stem cell of some CD34 and particular B cell subsets.P selects the albumen selective expression on activated blood platelet and endotheliocyte.P selects the interaction between albumen and PSGL-1 to promote the rolling of leukocyte on blood vessel wall, and the unusual gathering of leukocyte on vascular site causes various pathologic inflammation.The stereospecificity of last each tyrosine sulfuric ester of PSGL-1 selects albumen very important with combining of PSGL-1 for P.Electric charge is in conjunction with also very important: reduce NaCl (from 150 to 50mM) and strengthen in conjunction with (Kd～75nM).The sulphation of the last tyrosine of PSGL-1 strengthens PSGL-1 and sticks on the P selection albumen, but is not that adhesion is essential.PSGL-1 tyrosine sulphation all supports slower rolling to adhere under all shear rates, and supports the adhesion (Rodgers et al., Biophys.J.81:2001-09 (2001)) of rolling under higher shear rate.In addition, the someone to propose PSGL-1 be 25-100/one (Frenette et al., J.Exp.Med.191 (8): 1413-22 (2000)) who expresses on leukocyte in the expression on the platelet.

The anti-people PSGL-1 monoclonal antibody KPL1 of commercialization supply has shown can suppress PSGL-1 and P selects between albumen and PSGL-1 and L select interaction between protein.(Snapp etal., Blood 91 (1): 154-64 (1998)) in the tyrosine sulphation zone (YEYLDYD) of PSGL-1 for the KPL1 epitope mapping.

Also suppress the formation of tumor cell-platelet complex with O-sialoglycoprotein enzyme pretreatment tumor cell, sialylated and fucosylated mucin part has been removed in described processing.Experiment shows that in these treatments any causes combining of more mononuclear cells and circulating tumor cell in the body, show and reduce platelet in conjunction with impelling immunocyte contact circulating tumor cell (Varki and Varki, Braz.J.Biol.Res.34 (6): 711-17 (2001)).

Fibrinogen

2 kinds of normal person's Fibrinogens-normal γ type and γ ' type arranged, and every kind all can be found in normal individual.The normal fiber proteinogen is main (accounts in the body total fiber proteinogen about 90%), is made up of 2 identical 55kDa α chains, 2 identical 49.5kDa γ chains with 2 of identical 95kDa β chain.Normal variant Fibrinogen less (account in the body total fiber proteinogen about 10%) is made up of 2 identical 55kDa α chains, 2 identical 95kDa β chains, 1 49.5kDa γ chain and 1 variant 50.5kDa γ ' chain.γ chain and γ ' chain are encoded by homologous genes, by 3 ' end alternative splicing take place and form.Normal γ chain is made up of the 1-411 amino acids, and variant γ chain is made up of 427 aminoacid, and wherein the 1-407 amino acids is identical with normal γ chain and the 408-427 amino acids is VRPEHPAETEYDSLYPEDDL.This zone is occupied by the thrombin molecule usually.

Fibrinogen is converted into fibrin through thrombin action in the presence of ionized calcium, blood is produced condense.Fibrin also is the composition of thrombosis and acute inflammation exudate.

Purpose

One of the object of the invention provides antibody, its fragment or its complex, described antibody, its fragment or its complex combine with epi-position on the various molecules, described molecule relates to as processes such as cell rolling, inflammation, immunoreation, infection, autoimmune response and transfers, but do not relate to as adhesion, thrombosis and/or processes such as restenosis and gathering, described epi-position is present on the disease cell, as AML cell, T-ALL cell, Pre-B-ALL cell, B leukemia, B-CLL cell, multiple myeloma cells and transitional cell.

Another object of the present invention comprises the purposes of above-mentioned antibody in drug development and preparation, described medicine is used to suppress cell rolling, inflammation, immunoreation, infect, autoimmune response and transfer, but do not relate to adhesion, thrombosis and/or restenosis and gathering, this medicine also is used for the treatment of disease such as AML, T-ALL, the B leukemia, B-CLL, Pre-B-ALL, multiple myeloma, shift, cardiovascular disease such as myocardial infarction, retinal diseases, rely on the disease that the protein-protein interaction of sulphation tyrosine causes, or these cell functions or act on obvious other disease that acts on.

A further object of the invention is above-mentioned antibody in diagnosis, predict prognosis or the application in the method for various disease state individuality by stages, described disease such as AML, T-ALL, B leukemia, B-CLL, Pre-B-ALL, multiple myeloma and transfer, or cell function or effect as cell rolling, inflammation, immunoreation, infection, autoimmune response, transfer etc. play obvious other disease that acts on.Another object of the present invention provides the method for eliminating tumor cell.

Another object of the present invention provides by giving the method for described antibody activation ADCC or stimulation NK cell or T cell.

Above and other objects of the present invention are provided herein.

The invention summary

The invention provides antibody or its fragment with SEQ ID NO:1 scFv antibody fragment binding ability.The present invention also provides antibody or its fragment, and wherein at least a antibody or its binding fragment have first hypervariable region of SEQ ID NO:2, second hypervariable region of SEQ ID NO:3 and/or the 3rd hypervariable region of SEQ ID NO:4.Antibody of the present invention or its fragment preferably combine or cross reaction with the epi-position of PSGL-1.Antibody of the present invention or its fragment epi-position also preferred and at least a following cell combines, and described cell is selected from T-ALL, AML, B leukemia, B-CLL and multiple myeloma leukaemia.

The present invention also provides isolating epi-position, described epi-position have can with antibody of the present invention or the bonded aminoacid sequence of its binding fragment.Preferred this isolating epi-position is among the negative charge aminoacid bunch between 1 and 17 amino acids of ripe PSGL-1.

The present invention also provides above-mentioned antibody or its segmental Pharmaceutical composition and produces above-mentioned antibody or its segmental method.The present invention also provides the method with this Pharmaceutical composition treatment various diseases, and described disease comprises to relate to and suppresses or the treatment cell rolling; Suppress or the treatment inflammation; Suppress or the treatment autoimmune disease; Suppress or treatment infection (as viral infection such as HIV); Suppress or the treatment transfer; Suppress or treatment growth of tumour cell and/or propagation; Increase the death of neoplastic cells rate; Suppress leukaemia's growth and/duplicate; Increase leukaemia's mortality rate; Change the sensitivity of disease cell enantiopathy medicine damage; Increase the sensitivity of tumor cell to the anticarcinogen damage; Increase the sensitivity of leukaemia to the antileukemia damage; Suppress to suffer from the increase of tumor cell quantity among the patient of tumor; Reduce the quantity of tumor cell among the cancered patient; Suppress to suffer from the increase of leukaemia's quantity among the leukemic patient; Reduce the quantity of leukaemia among the leukemic patient.The present invention also provides other method that causes ADCC or stimulation NK cell or T cell with antibody of the present invention or its fragment.

The present invention also provides the method for eliminating the patient tumors cell, and described method is for providing the cell sample of taking from the patient, with patient's cell and antibody of the present invention or polypeptide incubation.

Definition

Antibody (Ab) or immunoglobulin (Ig) are and the bonded protein molecular of antigen.Each function bonding unit of natural generation antibody is the unit (2 heavy chains and 2 light chains) that forms by 4 polypeptide chains that disulfide bond connects.Every chain has constant region and variable region.Natural generation antibody can be divided into several classes based on its heavy chain composition, comprises IgG, IgM, IgA, IgD and IgE.The IgG class comprises some subclass, including but not limited to IgG ₁, IgG ₂, IgG ₃And IgG ₄Immunoglobulin is produced by bone-marrow-derived lymphocyte in vivo, and the specific exogenous antigen determinant of each this molecular recognition also promotes described antigenic removing.

Can and use antibody with many form productions, comprise antibody complex.Term used herein " antibody complex " is used in reference to the complex of one or more antibody and other 1 antibody or one or more antibody fragments, or the complex of 2 or a plurality of antibody fragments.The example of antibody fragment comprises Fv, Fab, F (ab ') ₂, Fc and Fd fragment.Accordingly, term used herein " antibody or its fragment " comprises antibody complex.

Described in this paper description and claim, Fv is defined as the molecule of being made up of human antibody heavy chain variable region and human antibody light chain variable region, described variable region can be identical or different, wherein variable region of heavy chain link to each other with variable region of light chain, in conjunction with, fusion or covalently bound.Fv can be strand Fv (scFv) or the stable Fv (dsFv) of disulfide bond.ScFv comprises the variable region of heavy chain of antibody and light chain, and the variable region is by base or the connection of connection base at interval of flexible amino acid polypeptide.Connecting base can be with side chain or not be with side chain.Preferred connection base is a 0-15 amino acid residue, and most preferably connecting base is (Gly ₄Ser) ₃

The Fv molecule itself comprises first chain and second chain, and every chain has first, second and the 3rd hypervariable region.Hypermutation ring in light chain and the variable region of heavy chain is called complementary determining region (CDR).In every heavy chain and the light chain CDR1, CDR2 and CDR3 district are arranged.These zones form antigen binding site, and can be modified to obtain enhanced binding ability especially.The most changeablely in essence in these zones be heavy chain CDR3 district.The CDR3 district is understood that the area exposed of Ig molecule, and the site for observed selectivity and/or specificity binding characteristic are played a major role as shown here.

The fragment of Fv molecule is defined as any less than original Fv but still keep the molecule of original Fv selectivity and/or specificity binding characteristic.This segmental example only comprises the small-sized antibody (minibody) of Fv heavy chain fragment including but not limited to (1), (2) comprise the miniantibody (international application no PCT/IL99/00581) of the small fragment unit of antibody heavy chain variable region, (3) has the similar antibody (similar bodies) of light chain segments, the similar antibody that has the variable region of light chain functional unit with (4).What should understand is that the Fv molecule fragment can be circular or annular polypeptide.

Term used herein " Fab fragment " is the monovalent antigen binding fragment of immunoglobulin.The Fab fragment is made up of light chain and part heavy chain.

F (ab ') ₂The bivalence Fab of fragment for obtaining by the pepsin digestion immunoglobulin.It comprises the part of 2 light chains and 2 heavy chains.

The Fc fragment is the non-antigen-binding portion thereof of immunoglobulin.It comprises heavy chain C end parts and Fc receptor binding site.

The Fd fragment is the immunoglobulin heavy chain variable region and first constant region.

Polyclonal antibody is the product of immunne response, is produced by some different B lymphocytes.Monoclonal antibody derives from single clone B cell.

The box that defines and be applied to polypeptide among the present invention is meant given continuous amino acid sequence, and it is considered to a unit as skeleton, can operate on it.Can replace, insert, lack or connect a plurality of aminoacid in its one or both ends.Equally, can replace, insert, lack or connect a plurality of aminoacid sections in its one or both ends.

Term used herein " epi-position " refers to antibody, antibody fragment, antibody complex or has complex or the interactional antigenic determinant of TXi Baoshouti or the recognition site or the antigen site of its binding fragment.Term epi-position used herein can be exchanged with term part, territory and calmodulin binding domain CaM.

The definition of herein selectivity is selected from entity or entity attitude mixture for target molecule and in conjunction with the ability of a kind of entity or cell, wherein all entities or entity attitude can have specificity to target molecule.

Term used herein " affinity " is to weigh between binding molecule (as a binding site on the antibody) and the part (as antigenic determinant) in conjunction with strength (binding constant).The strength summation of noncovalent interaction is the affinity of antibody to this epi-position between each antigen binding site on the antibody and each epi-position.Low-affinity antibody combines more weak with antigen and tendency is dissociated, and high affinity antibody combines tightr with antigen and keep the bonding state longer time.Term " avidity " is distinguished to some extent with affinity, because the former reflects the effectiveness of antigen-antibody interaction.

The specificity of antibody-AI: though antigen-antibody reaction is special, the antibody that produces with a kind of antigen under some situation can produce cross reaction with the irrelevant antigen of another kind.If 2 not synantigen have homology or analog structure, epi-position or its anchorage zone, if or specificity combine with irrelevant epi-position at the antibody of a certain epi-position with analog structure structure picture or chemical property, cross reaction promptly takes place.

Platelet is Megakaryocytic disc kytoplasm fragment, and it comes off in bone marrow sious, circulates in PBF subsequently.Platelet has some physiological function, is included in the main effect that plays in the blood coagulation.Platelet comprises granule and the periphery hyalomitome that is positioned at the center, but does not have clear and definite nuclear.

Coagulation used herein refers to that the particle adhesion of the antibacterial, cell, platelet or other the similar size that suspend forms the process of grumeleuse.This process and precipitated phase seemingly, but granule is bigger, mostly is suspension rather than solution.

Term is assembled and is referred to that the machine-processed continuously part of the common conduct of platelet and thrombin and collagen at the external caking that causes, forms thrombosis or tampon.

Conservative amino acid replacement is defined as the change that aminoacid is formed, and described change is undertaken by 1 or 2 aminoacid in conversion peptide, polypeptide or albumen or its fragment.This displacement is the aminoacid (as acidity, alkalescence, fragrant shape, size, plus or minus electric charge, polarity, nonpolar) with similar quality, therefore displacement can the material alterations peptide, polypeptide or proteic characteristic (as electric charge, isoelectric point, IP, affinity, avidity, conformation, dissolubility) or activity.General conservative amino acid replacement occurs in the following aminoacid group:

Glycine (G), alanine (A), valine (V), leucine (L) and isoleucine (I);

Aspartic acid (D) and glutamic acid (E);

Alanine (A), serine (S) and threonine (T);

Histidine (H), lysine (K) and arginine (R);

Agedoite (N) and glutamine (Q);

Phenylalanine (F), tyrosine (Y) and tryptophan (W).

Conservative amino acid replacement can carry out in lower area, as the hypervariable region flank region that molecular selectivity and/or specificity binding characteristic are played a major role, and the other parts of molecule such as variable heavy chain box.In addition, modification can be finished by rebuilding molecule, forming full-scale antibody, disome (dimer), trisome (trimer) and/or limbs (tetramer), or forms small-sized antibody or miniantibody.

Phasmid is defined as the phage particle that carries plasmid DNA.Phasmid contains the plasmid vector of filobactivirus replication origin such as fd phage m13 for design.Owing to carry plasmid DNA, phase granule does not have enough spaces to comprise a whole set of phage genome.The composition that lacks in the phage genome is the necessary information of assembling phage particle.Therefore, be the breeding phage, must be with target phage granule and the helper phage co-cultivation that can supply the assembling loss of learning.

Promoter is that DNA goes up RNA polymerase in conjunction with also starting the zone of transcribing.

Phage display library (being also referred to as phage display peptide/antibody library, phage library or peptide/antibody library) comprises a large amount of phagies (10 ⁸Or more), each phage particle is showed different peptide or peptide sequence.These peptides or polypeptide fragment can be configured to all lengths.The peptide of showing or the source of polypeptide are including but not limited to human antibody heavy chain or light chain.

Pharmaceutical composition refers to comprise the preparation of antibody of the present invention or peptide or polypeptide and pharmaceutical acceptable carrier, excipient or diluent, or refers to antibody-drug complex and its pharmaceutical acceptable carrier, excipient or diluent.

Medicine refer to be used for to the mammal active disease treat, the medicine of prophylactic treatment or diagnosis, mammal is including but not limited to people, cattle, horse, pig, mice, dog, cat or any other homoiothermic animal.Medicine is selected from radiosiotope, toxin, medical substance, oligonucleotide, recombiant protein, antibody fragment, anticarcinogen, anti-adhesive medicine, antithrombotic drug, anti-restenosis, anti-autoimmune medicine, anti-medicine, antimicrobial drug, antiviral agents and the anti-inflammatory agent assembled.Other example of these medicines is including but not limited to the antiviral agents that comprises acyclovir, ganciclovir and zidovudine; Antithrombotic formation/the restenosis that comprises cilostazol, dalteparin sodium, Reviparin Sodium and aspirin; Comprise zaltoprofen, pranoprofen, drogelor, aspirin 17, diclofenac, ibuprofen, dexibuprofen, sulindac, naproxen, ammonia tolmetin, match the anti-inflammatory agent of examining former times, indomethacin, rofecoxib and nimesulide; The anti-autoimmune medicine that comprises leflunomide, denileukin diftitox, subreum, WinRho SDF, defibrotide and cyclophosphamide; And comprise limaprost, cloricromen and hyaluronic anti-adhesive/anti-medicine of assembling.

Antileukemia is the medicine with anti-leukocythemia liveness.For example, antileukemia comprises the medicine that suppresses or stop leukaemia or the growth of immaturity Preleukemia cell, kill the medicine of leukaemia or Preleukemia cell, increase leukaemia or Preleukemia cell medicine, and suppress the medicine that the leukaemia shifts other antileukemia sensitivity.In the present invention, antileukemia also can be prevention, suppresses, delays or stops the anti-angiogenesis activity medicine that tumor vessel forms.

To the research of expression of gene pattern can by analyze under various conditions, special time, the various amount of the gene expression product of medium generation of organizing carry out.When the amount of gene outcome is higher than the amount of finding, think that gene is " overexpression " in normal control such as non-morbid state contrast.

Certain cell can have the albumen of given antibody combining site (or epi-position) at its surface expression, but cell in the stage binding site can hidden form exist (as sterically hindered or sealing, or lack the binding antibody desirable characteristics), this is called as the first phase (I phase).The I phase can be normally, healthy, non-disease conditions.When epi-position existed with hidden form, its not given antibody recognition did not promptly have antibody to combine with this epi-position or given cell of I phase.But, this epi-position can owing to itself experience modify or because near it or continuous molecule is modified or since certain zone experience structure picture change expose.The example of modifying comprises folding change, the change of post translational modification, the change of phospholipidization, Sulfated change, glycosylated change etc.These changes can take place when cell enters different phase, and this is called as the second phase (II phase).The example of the second phase comprises activation, breeds, transforms or is in malignant state.Through modifying, epi-position exposes, and antibody can combine with it.

Peptide mimics is not comprise the molecule that any peptide bond is an amido link (as antibody) between the aminoacid; But in content of the present invention, the term peptide mimics comprises not exclusively and is the molecule of native peptides, as pseudo-peptide, half peptide and class peptide.No matter be to be non-peptide wholly or in part, peptide mimics of the present invention provides steric reactive chemical part, with very similar as the three-dimensional arrangement of active group in the peptide on peptide mimics basis.These molecules comprise micromolecule, fat, polysaccharide or its conjugate.

The accompanying drawing summary

Fig. 1 represents the facs analysis after the T-ALL cell dyes with TM3.13 scFv.

Fig. 2 represents to come comfortable scFv antibody to have the numeric data of the accumulative Lumiaggregometer analysis of platelet down, is expressed as washing platelet or PRP and contrasts accumulative percentage ratio.

Fig. 3 represents more various scFv antibody and the bonded facs analysis of platelet: Fig. 3 A is AN51-PE (FSC), and Fig. 3 B is AN51-PE (FL2-H), the negative contrast of Fig. 3 C, and Fig. 3 D is Y1-myc+, and Fig. 3 E is Y1, and Fig. 3 F is L32, and Fig. 3 G is TM1.1.

Fig. 4 represents comparison variable concentrations scFv antibody (N01, Y1-myc+ and L32) competition and disturbs labelling Y1 antibody and the bonded facs analysis of KG-1 cell: Fig. 4 A is 0ng, and Fig. 4 B is 100ng, and Fig. 4 C is 250ng, Fig. 4 D is 500ng, Fig. 4 E is 1000ng, and figure F is 2500ng, and Fig. 4 G is 5000ng.

Fig. 5 represents comparison scFv antibody (N01, Y1-myc+ and L32) competition and disturbs the numeric data of labelling Y1 antibody and the bonded facs analysis of KG-1 cell.

Fig. 6 represents from the numeric data of variable concentrations scFv antibody (TM1.1, Y1-myc+, Y1 and L32) with sugar small cup protein binding elisa assay relatively is provided.

Fig. 7 represents L32 and Y1 scFv antibody and GC, blood plasma and KG-1 and the bonded Western analysis of Raji epicyte protein.

Fig. 8 represents to provide the numeric data of variable concentrations scFv antibody (Y1-myc+, TM1.1 and L32) with Fibrinogen, PSGL-1 and GPIb α related peptides binding ratio elisa assay.

Fig. 9 is illustrated in the numeric data of the facs analysis after various concentration GPIb derived peptide exist following platelet with Y17 scFv antibody staining.The result represents with single geometrical mean form with scFv antibody gained reaction reduction percentage ratio.

Figure 10 represents that Y1 and Y17 scFv antibody combine the numeric data of back elisa assay with the Sulfated PSGL1 of various diverse locations.

Figure 11 represents the numeric data of facs analysis, and relatively scFv antibody (Figure 11 A is TM1.1, and Figure 11 B is TM1.3, and Figure 11 C is L32) and T-ALL cell and normal circumference hemocyte (N-PBL) combines variation with respect to each scFv antibody concentration.

Figure 12 represents to give L32 and the numeric data of Y1 scFv antibody to studying in the effect body of liver weight (Figure 12 A) and tumor incidence rate (Figure 12 B) in the SCID mice Molt4 cell tumour model.

Detailed Description Of The Invention

The present invention relates to have antibody or its fragment of SEQ ID NO:1 scFv antibody fragment in conjunction with the PSGL-1 ability.Therefore, these antibody of the present invention have the similar binding affinity with SEQ ID NO:1.The scFv fragment of SEQ ID NO:1 is called as L32.Accordingly, preferred antibody of the present invention is L32.This antibody only has multifarious phage library to determine by screening in heavy chain CDR3 district, select the specific antibody of identification leukaemia surface antigen determinant, the wherein unknown in advance or not evaluation of specific receptor by the leukemia cell.Adopting uses the same method has identified another antibody L31.Though the present invention comprises many antibody, L32 is exemplary to be used for hereinafter.

In the past at U. S. application number 10/032,423; 10/032,037; 10/029,988; 10/029,926; 09/751,181 and 60/258,948 and international application no PCT/US01/49442 and PCT/US01/49440 in use same phage library to determine other and the bonded antibody of leukaemia.The instantiation of disclosed antibody comprises Y1 and Y17 antibody in these applications.Find that the epitope specificity that exists on disclosed antibody and the hematopoietic cell protein in these applications combines, described epi-position N holds tyrosine by sulphation, is considered to relate to cell migration, as neoplasm metastasis.

Disclosed antibody all combines with the leukaemia in L32 antibody and the Y1/Y17 application, though L32 and the bonded affinity of leukaemia than Y1 about 5 times.Based on this fact, and the antibody separation compares research to determine them separately in conjunction with the dependency between the epi-position from common species system (DP32) this fact.Confirm the bonded sulphation epi-position of L32 subsequently and propose its expression, L32 is estimated with hematoblastic the combination also than low 25-100 times (Frenette et al., J.Exp.Med.191 (8): 1413-22 (2000)) on leukocyte.But find that L32 only combines with platelet is insignificant, and do not influence platelet aggregation.That lists in the table 1 is the scFv of Y1 and the IgG summary than scFv and the IgG of L32.

Table 1

	??Y1?scFv	??Y1?IgG	?L32?scFv	?L32?IgG
	??Y1?scFv	??Y1?IgG	?L32?scFv	?L32?IgG	WBC in conjunction with the leukaemia in conjunction with the reactive platelet aggregation GPIb reactivity kytoplasm composition in vitro effects of being combined with the competitive blood platelet of KPL1 of PSGL-1	Low lower high binding fiber proteinogen γ ' CCF4 that suppress do not measure	Gao Gaogao is that height is induced binding fiber proteinogen γ ' CCF4 ADCC	Gao Gaogao be low-do not have not have impact very low-do not have and do not measure	Very high very high be low-do not have-very low-do not have very low-no ADCC

That identifies previously exists the sulphation part with being characterized as of the bonded sulphation epi-position of Y1/Y17, as sulphation tyrosine residue or sulphation saccharide or fat part, preferably at 2 or more in the amino acids bunch, described sulphation partly is found on part and the receptor, plays an important role in various process such as inflammation, immunoreation, infection, autoimmune response, transfer, adhesion, thrombosis and/or restenosis, cell rolling and gathering.These epi-positions are also found on the disease cell, as B leukaemia, B-CLL cell, AML cell, multiple myeloma cells and transitional cell.These epi-positions can be used for comprising by stages diagnosis or predict prognosis method for the useful target of these processes of treatment.

L32 scFv is stronger to sulphation PSGL-1 selectivity.In disease such as atherosclerotic inflammatory process, in inflammatory leukocytes such as mononuclear cell, neutrophil(e) cell and lymphocyte master's the inflammatory process, inflammatory leukocytes such as mononuclear cell, neutrophil(e) cell and lymphocyte are mainly raised (Huoand Ley by 4 kinds of adhesion molecules (PSGL-1, P select albumen, VLA-4 and VCAM-1), Acta Physiol.Scand., 173:35-43 (2001); Libby, Sci.Am.May:48-55 (2002); Wang et al., J.Am.Coll.Cardiol.38:577-582 (2001)).L32 is to the latent effect of interference prompting L32 in eliminating relevant disease of these important molecule.Specifically, P selects protein regulation cell adhesion and rolling.In addition, P selects the interaction of albumen and PSGL-1 to activate on some cells and produces (when relating to malignant cell) and whole relevant other molecule (Shebuski andKilgore, J.Pharmacol.Exp.Ther.300:729-735 (2002)) of inflammatory reaction (when relating to leukocyte) with tumor.Based on this understanding of P being selected protein regulation cell processes ability, obviously L32 scFv can make it more have superiority as various pernicious and molecules diseases associated with inflammation of treatment to the stronger selectivity of sulphation PSGL-1.In addition, the model of malignant disease shows that P selects albumen and combining of malignant cell to need the sulphation (Ma and Geng, J.Immunol.168:1690-1696 (2002)) of PSGL-1.This requirement to L32 bonded require similar.Therefore can estimate that L32 can eliminate P and select the promotion of albumen to carrying out property malignant disease.

In addition, also find antibody of the present invention in conjunction with dependent cells stage of development (AML hypotype use under conventional method and cytochemical staining observed morphology based on the French-American-British system divides): antibody and M3 hypotype or above AML cell combine, but do not combine with M0 or M1 hypotype cell.In addition, antibody can combine with M2 hypotype cell or can not in conjunction with.Accordingly, antibody of the present invention does not combine with normal health medullary cell (as the CD34+ cell).These differences are considered to express and/or Sulfated change based on PSGL-1, and the structure picture of possible a little the different epi-position of exposure of PSGL-1 changes.

Preferred L32 antibody of the present invention and different molecule or the epi-position combinations that relate to inflammation are as PSGL-1.Also preferred L32 antibody combines with at least a epi-position that exists on inflammation or the tumorigenic cell that relates to, and comprises T-ALL cell, AML cell and B leukaemia.L32 antibody more preferably of the present invention combines with epi-position on fat, saccharide, peptide, glycolipid, glycoprotein, lipoprotein and/or the lipopolysaccharide molecule.Described epi-position preferably has at least one sulphation part.Perhaps but also preferred L32 antibody and two or more epi-position cross reaction, every kind of epi-position have one or more sulphation tyrosine residues with at least one 2 or more the polyacid acidic amino acid bunch, one of them example is PSGL-1.

With after the PSGL-1 that exists on the cell surface combines, but some antibody of the present invention or its fragment internalization enter cell.But general complete IgG antibody internalization, and less antibody fragment (as scFv) can not internalization.Should be understood that but the antibody internalization enters the cell of any expression PSGL-1, for example comprises the AML cell.Internalization can take place by endocytosis, and described endocytosis is the active process that mode, time and temperature rely on.

The hypervariable region of L32 antibody of the present invention participates in the formation of antigen binding site.The complementary structure of antigen binding site and antibodies epi-position, so these binding sites are called complementary determining region (CDR).3 CDR (CDR1, CDR2 and CDR3) are arranged on every light chain of antibody and the heavy chain, and each is positioned at and V _HAnd V _LOn the ring that domain β lamella links to each other.The most variable in these zones is heavy chain CDR3 district.The CDR3 district is considered to the area exposed of Ig molecule, as described hereinly plays central role in observed selectivity of decision and/or specificity binding characteristic.

DP32 is that phage display library one of is for 49 kinds, is that a kind of phage library distinct species is, scFv antibody of the present invention separates acquisition by it.Therefore, DP32 provides heavy chain and light chain skeleton variable region, light chain CDR1, CDR2 and CDR3 district at least for antibody of the present invention, and/or heavy chain CDR1 and CDR2.DP32 also provides the three dimensional structure consistent with the hypervariable region.The specificity of antibody is known by its three-dimensional structure picture decision.Therefore, the restriction that applies of DP32 can play remarkable effect in decision L32 antibody specificity.In addition, DP32 has multiple electric charge aminoacid, and this also plays structural effect in the identification of L32 antibody.

According to the present invention, CDR can be inserted into and produce antibody in the box.Definition and the box that is applied to polypeptide refer to the continuous amino acid given sequence as skeleton among the present invention, are considered to a unit, can operate on it.Can replace, insert, lack or connect a plurality of aminoacid in its one or both ends.Equally, can or connect the aminoacid section in its one or both ends displacement, insertion, disappearance.

The aminoacid sequence of box is fixed substantially, but displacement, insertion or catenation sequence are alterable heights.Box can comprise some zone, and each zone comprises final formation thing important function.

The box of specific embodiments of the present invention comprises skeleton district 1 (FR1), CDR1, skeleton district 2 (FR2), CDR2, skeleton district 3 (FR3) and skeleton district 4 (FR4) from the N end.

In an embodiment of the present invention, can in box, replace significantly district.For example, the CDR2 of box and CDR1 hypervariable region can be replaced by non-conservative, preferred conserved amino acid or modify.

In a preferred embodiment of the present invention, antibody or its fragment have heavy chain and light chain, and every chain has first, second and the 3rd hypervariable region, is respectively CDR3, CDR2 and CDR1 district.Specifically, the CDR3 district of a chain determines in conjunction with selectivity and specificity, promptly light chain CDR3 district, be preferably heavy chain CDR3 district, the CDR3 district of heavy chain and light chain more preferably.Secondly, determine in conjunction with selectivity and specificity CDR2 and CDR1 district by light chain and heavy chain (preferably).First, second and/or flank upstream, the 3rd hypervariable region or downstream area also can influence in conjunction with selectivity and specificity.

In a preferred embodiment of the present invention, at least a antibody or its fragment have first hypervariable region (CDR3) of SEQID NO:2.In addition, at least a antibody or its fragment have second hypervariable region (CDR2) of SEQID NO:3.In addition, at least a antibody or its fragment have the 3rd hypervariable region (CDR1) of SEQID NO:4.More preferably at least a antibody or its fragment have first hypervariable region (CDR3) and second hypervariable region (CDR2) of SEQ ID NO:3 and the 3rd hypervariable region (CDR1) of SEQ ID NO:4 of SEQ ID NO:2.

In a concrete preferred embodiment, at least a antibody or antibodies fragment are the scFv with SEQ ID NO:1.

The aminoacid sequence (as CDR district, CDR flanking region) that owns≤25 amino acid residues for this paper description and detailed description, should understand and think that the further embodiment of the present invention is to comprise 1 or 2 aminoacid replacement in these aminoacid sequences, described replacement is preferably conserved amino acid and replaces.All aminoacid sequences for this paper description and detailed description greater than 25 amino acid residues, should understand and think that embodiment of the present invention is the aminoacid sequence (Altschul et al., Nucleic AcidsRes.25:3389-402 (1997)) that comprises in the scope of these aminoacid sequences with former sequence similarity 〉=90%.Similar or homologous amino acid is defined as the different aminoacids with similar quality, and is similar as acidity, alkalescence, aromatics, size, positive charge or negative charge, polarity, non-polar nature.

Amino acid similarity or homology or sequence similarity percentage ratio are by comparing the decision of 2 kinds of different peptides or amino acid sequence of polypeptide.Antibody sequence is determined by dna sequencing.Usually use a kind of computer program that is designed for this purpose, arrange 2 sections sequences, at each position comparing amino acid residue.Determine amino acid whose concordance or homology then.Utilize a kind of algorithm to determine amino acid similarity percentage ratio then.Usually preferred comparing amino acid sequence, this is owing to the sensitivity of measuring exact relationship between peptide, polypeptide or the protein molecular has strengthened greatly.The albumen comparison can consider that conserved amino acid replaces, because if different aminoacids is when having similar physics and/or chemical property, mispairing can just produce and divide (Altschul et al. (1997), the same).

In one embodiment of the invention, 3 hypervariable regions of each light chain and heavy chain can exchange between 2 chains and in the chain and/or between interchain 3 hypervariable sites.In addition, can change the sequence of hypervariable region to cross over 2 or more CDR.Can occur in equally in the skeleton variable region, also can only partly occur in 1 CDR.

The invention provides peptide or polypeptide with antibody or its Fab or its construction or its fragment construction.According to the present invention, antibody comprises IgG, IgA, IgD, IgE or IgM antibody.The IgG class comprises some hypotypes, comprises IgG ₁, IgG ₂, IgG ₃And IgG ₄

Antibody can provide by various forms, as fragment, complex and polymer.According to the present invention, antibody fragment comprises Fv, scFv, dsFv, Fab, Fab ₂With the Fd molecule.Less antibody fragment as Fv fragment and Fab fragment, is also contained in the term " fragment ", as long as they keep original antibody or bigger segmental binding characteristic.Construction comprises for example polymer of disome, trisome and limbs.Except as otherwise noted or based on context and/or this area knowledge show, phrase " antibody; its binding fragment; or have the complex of antibody or its binding fragment " and " antibody or fragment " comprise all these molecules, with and derivant, compositions, trim, homologue, analogies and variant thereof.

It has been determined that scFv can pass tissue, because its size is less, its speed of removing from blood is than full size antibody fast (Adams et al., Br.J Cancer 77:1405-12 (1988); Hudson, Curr.Opin.Immunol.11 (5): 548-557 (1999); Wu etal., Tumor Targeting 4:47 (1999)).Therefore, scFv is usually used in relating to radiolabeled diagnosis such as tumor imaging, makes that radioactive label is faster to be removed in body.Some cancer targeting scFv polymer recently carry out clinical precursor internal stability and effect evaluation (Adamset al. (1988), the same; Wu (1999), the same).

General scFv monolithic design is V _HThe C end in territory is by polypeptide catenation sequence and V _LThe N end residue in territory links to each other.Optional rightabout: the V that uses _LThe C end in territory is by polypeptide catenation sequence and V _HThe N end residue continuous (Power et al., J.Immun.Meth.242:193-204 (2000)) in territory.The general length of polypeptide catenation sequence is about 15 aminoacid.When catenation sequence was reduced to 3-7 aminoacid, scFv can not be folded into the Fv territory of function, perhaps linked to each other with second scFv to form disome.Catenation sequence length further is reduced by at least when 3 aminoacid, according to catenation sequence length, composition and source, Fv territory, forces scFv to be connected to trisome or limbs (Powers (2000), the same).

Recent findings, multivalent antibody fragment such as scFv disome, trisome or limbs often provide above its parental generation antibody and the bonded affinity of target.This high affinity provides potential advantage, comprises strengthening the pharmacokinetics that cancer target is used.In addition, when the P that relates to leukocyte adhesion and rolling in research selected albumen and part PSGL-1 thereof, scientist infers the cell of expressing the PSGL-1 dimeric forms because its high binding affinity shows more stable rolling adheres to.This adhesion is more high to the shearing force repellence, shows littler rolling rate change (Ramachandran et al., PNAS, 98 (18): 10166-71 (2001)).

ScFv multivalence form is by other people's design and production.A kind of method is to connect 2 scFv with catenation sequence.Another kind method relates to uses disulfide bond to connect between 2 seFv.Produce dimer or the simplest method of trimer Fv by Holliger et al., PNAS 90:6444-48 (1993) and Kortt et al., Protein Eng.10:423-33 (1997) report.A kind of dimeric method of preparation scFv that is designed for is to add FOS and JUN protein region sequence at the C of scFv end, forms leucine zipper (Kostelny et al., J Immunol.148 (5): 1547-53 (1992) between them; De Kruif et al., J Biol Chem.271 (13): 7630-34 (1996)).It is to add the plain sequence of strepto-affinity at the C of scFv end that another kind is designed for the tetrameric method of preparation.Strepto-affinity element is made up of 4 subunits, and therefore when scFv-strepto-affinity element was folding, 4 subunits form the tetramers, and (Kipriyanov et al., Hum AntibodyHybridomas 6 (3): 93-101 (1995)).Another kind of preparation dimer, trimer or tetrameric method are to introduce free cysteine in desirable proteins.Have the maleimide groups of various quantity (2-4) based on the crosslinked sequence of peptide, be used for that crosslinked (Cochran et al., Immunity 12 (3): 241-50 (2000)) with desirable proteins and free cysteine.

The binding affinity that these multivalence forms are higher helps diagnosis, predict prognosis, by stages and therapeutic scheme.For example, scFv can be used as and the bonded blocking drugs of target recipient, therefore the combination of blocking-up " natural " part.In this case, the high affinity between scFv and receptor to reduce dissociated probability, causes native ligand can not effectively combine with target in conjunction with being ideal.In addition, relate in target recipient and adhere to and roll or when target recipient was present on the cell in high shear force blood flow district such as the platelet, this high affinity was favourable.

In this system, phage library (as discussed herein above) can be designed for shows scFv, and described scFv is foldable to the unit price form in antibody Fv district.In addition, also as the above discussion of this paper, the suitable bacterial expression that is used for of construction.Genetic engineering scFv comprises heavy chain and variable region of light chain, by 15 amino acid whose flexible peptides base connections at interval of continuous programming code.The preferred interval base is (Gly ₄Ser) ₃This basic at interval length provides a little interval base with its aminoacid together, allows V _HAnd V _LThe district is folded into can have a function Fv territory with its target effective is bonded.

The variation of basic length is the another kind of method for optimizing that forms dimer, trimer and the tetramer (this area refers to disome, trisome and limbs usually respectively) at interval.Dimer foreshortens at the interval base that connects 2 scFv variable chains under the condition of a common 5-12 amino acid residue and forms.The interval base of this shortening stops the Fv territory that is folded to form function with 2 variable chains of a part.As an alternative, the complementary territory pairing of another molecule of territory is forced in the territory, produces 2 in conjunction with the territory.In a preferred method, 5 aminoacid (Gly are only arranged ₄Ser) interval base is used to constitute disome.This dimer can form from 2 identical scFv, or forms from 2 different scFv, and the selectivity and/or the specificity that keep parental generation scFv strengthen binding ability, and/or show enhanced adhesion or affinity.

In a similar manner, trimer foreshortens under the condition that is less than 5 amino acid residues usually at the interval base that connects 2 scFv variable chains and forms, and prevents to be folded to form with 2 variable chains of a part the Fv territory of function.As an alternative, 3 independent scFv molecules are united the formation trimer.In a method for optimizing, obtain trisome by removing this flexible spacer.Trisome can form from 3 identical scFv, or forms from 2 or 3 different scFv, and the selectivity and/or the specificity that keep parental generation scFv strengthen binding ability, and/or show enhanced adhesion or affinity.

Limbs foreshorten under the conditions of similarity that is less than 5 amino acid residues usually at the interval base that connects 2 scFv variable chains and form, and prevent to be folded to form with 2 variable chains of a part the Fv territory of function.As an alternative, 4 independent scFv molecules are united the formation tetramer.Limbs can form from 4 identical scFv, or form from 1-4 different scFv, and should keep selectivity and/or the specificity enhancing binding ability of parental generation scFv, and/or show enhanced adhesion or affinity.Base is less than under the situation of 5 amino acid residue length usually at the interval, whether forms aminoacid sequence and reaction condition that trisome or limbs rely on concrete scFv in the mixture.

In case pick out or develop antibody, fragment and construction with required binding ability, use guidance provided herein, in those skilled in the art's limit of power, can produce the construction and the fragment that keep original antibody characteristic.For example, can prepare complete antibody molecule, Fv fragment, Fab fragment, Fab ₂Fragment, dimer, trimer and other construction, they have kept the desirable characteristics of original antibody, fragment and the construction of selecting or developing.

Substituted amino acid but still keep antibody or segmental characteristic can carry out conserved amino acid and replace in those skilled in the art's limit of power if desired.But antagonist or fragment are modified, and as being connected with various medicines, and do not change its binding characteristic.But antagonist or fragment are carried out other and are modified, and as for producing the modification that stabilization of antibodies more or fragment are carried out, and do not change its specificity.For example, can carry out that the class peptide is modified, half class peptide is modified, cyclic peptide is modified, N is terminal modified, C is terminal modified, peptide bond modification, backbone modification and residue modify.Under the guidance of this description, in those skilled in the art's limit of power, also can test modified antibodies or fragment and whether change with the binding characteristic of evaluating them.

Same, use guidance provided herein, in those skilled in the art's limit of power, can change the binding characteristic of antibody, fragment or construction, to obtain molecule with better characteristic.For example, in case identify antibody with required character, can use at random or site directed mutagenesis producing various antibody variation bodies, and can screen these variants to obtain ideal characterisitics.

Use traditional method known in the art, the technical staff also can determine antibody or its fragment that other has the L32scFv binding ability.For example, other antibody can separate with biological elutriator described herein, wherein saidly is used to screen special phage display library with bonded molecule of L32 or cell, especially derives from leukemia, lymphoma and myeloma patient's library.

Antibody of the present invention also can have the labelling that can insert or be connected with fragment, and described being marked with helps its preparation and evaluation, can be used for diagnosis or predict prognosis, comprises by stages.Labelling can be removed from molecule subsequently.The example of useful labelling comprises AU1, AU5, BTag, c-myc, FLAG, glutamic acid-glutamic acid, HA, His6, HSV, HTTPHH, IRS, KT3, PROTEIN C, S-TAG , T7, V5 and VSV-G (Jarvik and Telmer, Ann.Rev.Gen., 32,601-18 (1998)).Labelling is preferably c-myc or KAK.

Antibody, its antibody fragment or construction, peptide, polypeptide, albumen and their fragment and construction can be produced in protokaryon or eukaryotic expression system.Production antibody and segmental method are well known in the art in protokaryon or the eukaryotic system.

As the discussion of the present invention's definition and this paper, eukaryotic cell system refers to produce peptide or polypeptide expression system by genetic engineering method, and wherein host cell is an eukaryotic cell.Eukaryotic expression system can be mammlian system, and peptide that produces in the mammalian expression systems or polypeptide preferably are substantially free of the mammal pollutant behind purification.The useful example of other eukaryotic expression system comprises yeast expression system.

The prokaryotic system that is used for production peptide of the present invention or polypeptide preferably uses the host of escherichia coli (E.Coli.) as expression vector.Peptide of producing in the escherichia coli system or polypeptide are substantially free of e. coli contamination albumen behind purification.Use prokaryotic expression system can cause some or all sequence N end provided by the invention to add methionine residues.As known in the art, can remove N end methionine residues after producing peptide or polypeptide, make the expression fully of peptide or polypeptide, an example is to use Aeromonas (Aeromonas) aminopeptidase (U.S. Patent number 5,763,215) under appropraite condition.

In a preferred embodiment of the present invention, produce antibody or its segmental method and have following steps: (a) obtain phage display library; (b) obtain molecule or cell, described molecule or cell can combine with antibody or its fragment of the scFv antibody fragment binding ability with SEQ ID NO:1; (c) the screening phage display library is to obtain the phage particle of displaying and molecule or bonded oligopeptide of cell or polypeptide; (d) produce antibody or its fragment, comprise at least a antibody or its binding fragment and also comprise can with molecule or bonded peptide of cell or polypeptide.

Antibody of the present invention and polypeptide can be compound with various medical substances (as in conjunction with, associating, merge or be connected), as medicine, toxin and radiosiotope, the optional effective carrier of pharmacy in addition forms the peptide-drug compositions that comprises antibody/polypeptide and disease-resistant and/or active anticancer medical substance.Said composition can be used for diagnostic purpose.

Be used for carrier example of the present invention and comprise glucosan, HPMA (a kind of hydrophilic polymer) or other polymer such as hydrophilic polymer, with and derivant, conjugate and trim.Perhaps, can use modified liposome, as the liposome (as Doxil, a kind of liposome that comprises a large amount of doxorubicins of commercialized supply) of scFv Y1 molecular modification.Can prepare this liposome, make it comprise a kind or multiple required medicine and mix, so that higher medicine/antibody ratio to be provided with antibody of the present invention.

Perhaps, the connection between antibody or its fragment and the medicine can be direct connection.Can be by the direct connection between 2 of generations of the chemical bond between element in the molecule or the element group or the more heterogeneous adjacent molecule.Chemical bond can be, for example, and ionic bond, covalent bond, hydrophobic bond, hydrophilic bond, electrostatic bond or hydrogen bond.Key of the present invention can be, for example, and amido link, carbon-sulfide linkage, peptide bond and/or disulfide bond.For antibody is linked to each other with medicine or catenation sequence, can use amine, carboxyl, hydroxyl, sulfydryl and ester function group, form covalent bond with technology known in the art.

Between peptide and the medicine or between peptide and the carrier or the connection between carrier and the medicine can finish by connecting chemical compound.The chemical compound that is connected that uses in this paper description and the claim is defined as and connects 2 or more part of chemical compound.Connect chemical compound and can be straight or branched.Side chain connects chemical compound can comprise two branches, three branches, four branches or the chemical compound of multiple-limb more.Be used for connection chemical compound of the present invention and can be selected from dicarboxylic acids, maleimide base hydrazides (malemido hydrazides), PDPH, carboxylic hydrazides and little peptide.

Be used for connection chemical compound of the present invention more specifically example comprise: (a) dicarboxylic acids such as succinic acid, 1,3-propanedicarboxylic acid and adipic acid; (b) maleimide base hydrazides such as N-[maleimide base caproic acid] hydrazides, 4-[N-maleimide ylmethyl] cyclohexyl-1-carboxylic hydrazides and N-[maleimide base undecanoic acid] hydrazides; (c) (3-[2-pyridine radicals two sulfur] the propionyl hydrazine); (d) be selected from carboxylic hydrazides and derivant, conjugate, trim and the analog of 2-5 carbon atom.

Also can use little peptide to connect chemical compound connects by direct coupling.For example the direct coupling of anticarcinogen doxorubicin free sugar and scFv is connected available little peptide and finishes.The example of little peptide comprises AU1, AU5, BTag, c-myc, FLAG, glutamic acid-glutamic acid, HA, His6, HSV, HTTPHH, IRS, KT3, PROTEIN C, S-TAG , T7, V5, VSV-G and KAK.

Antibody of the present invention and fragment thereof can with preparation (being also referred to as indicative labelling) (as radiosiotope) in conjunction with, be connected, compound or otherwise associating, described junctional complex can be used for diagnosis, predict prognosis, by stages or the imaging purpose.Test kit with radiosiotope-antibody (or fragment) junctional complex can be provided.

Be used to diagnose, predict prognosis or radioisotopic example by stages comprise ¹¹¹Indium, ¹¹³Indium, ^99mRhenium, ¹⁰⁵Rhenium, ¹⁰¹Rhenium, ^99mTechnetium, ^121mTellurium, ^122mTellurium, ^125mTellurium, ¹⁶⁵Thulium, ¹⁶⁷Thulium, ¹⁶⁸Thulium, ¹²³Iodine, ¹²⁶Iodine, ¹³¹Iodine, ¹³³Iodine, ^81mKrypton, ³³Xenon, ⁹⁰Yttrium, ²¹³Bismuth, ⁷⁷Bromine, ¹⁸Fluorine, ⁹⁵Ruthenium, ⁹⁷Ruthenium, ¹⁰³Ruthenium, ¹⁰⁵Ruthenium, ¹⁰⁷Hydrargyrum, ²⁰³Hydrargyrum, ⁶⁷Gallium and ⁶⁸Gallium.Preferred radiosiotope is transmitted X-rays or any paramagnetic ion not.

Indicative labelled molecule also can be fluorescent tag molecule.The example of fluorescent tag molecule comprises fluorescein, phycoerythrin or brilliant pink b, or its trim or junctional complex.

Antibody that is connected with indicative labelled molecule or fragment can be used for diagnosis, predict prognosis or by stages, by the sample that contains patient's cell is provided, determine that whether antibody of the present invention combines with patient's cell, shows the dangerous trouble of patient thus or suffers from disease.In addition, the present invention also provides the method for eliminating the patient tumors cell, and described method is by providing the sample that comprises patient's cell, and with patient's cell and antibody incubation of the present invention.These activities can be in vivo, external or stripped carrying out.When in vivo or exsomatize when carrying out, it is acceptable that preparation is preferably the physiology, promptly its can patient harm to unacceptable degree.Acceptable extent of injury can be by the clinicist according to the decision of standards such as disease severity and other drug candidate.

For cancer, patient disease by stages usually according to size, type, position and tumor invasion decision classification of diseases.A kind of is " classification of TNM malignant tumor " (6th Edition) (L.H.Sobin by the tumor feature to the system of cancer classification, Ed.), described system quotes by integral body and is attached to herein, this system is divided into T, N and M three classes according to cancer size and position with it, T represents primary tumo(u)r, N represents regional nodes, and M represents metastasis.In addition, digital I, II, III and IV are used for the expression phase, and each numeral refers to the combination that the TNM factor is possible.For example, the I primary breast cancer by the TMN class definition is: T1, and N0, M0, this expression: the T1-diameter of tumor is 2cm or littler, and N0-does not have metastases in local lymph node, and M0-does not have metastasis.Another system is used for AML by stages, uses under conventional treatment and cytochemical staining observed form according to French-American-British system divides hypotype.

In addition, The World Health Organization's suggestion recently to hemopoietic tissue and lymthoma disease by stages or classification comprise (especially for AMLS) traditional F AB type classification of diseases, and relate to other disease type and the AML relevant that special cells heredity is found with myeloproliferative disorder.Also has the pathology classification of other people's suggestion.For example, a kind of special suggestion at AML comprises the disease type that relates to special cells heredity transposition, can discern reliably by morphology evaluation and immunophenotype, and described classification has embodied the importance of relevant myelosis abnormal change.This system obtains cytogenetics or molecular genetic research is supported, and along with the appearance of describing the new clinical pathology discerned example also can be expanded (Arber, Am.J.Clin.Pathol.115 (4): 552-60 (2001)).

The invention provides diagnosis, predict prognosis or test kit by stages, be used for the treatment of during preceding, the treatment or the analyzed in vitro of treatment back therapeutic effect, described test kit has the peptide of the present invention that links to each other with indicative labelled molecule or preparation.The present invention further provides and use preparation cancer, the tumor of saying so more specifically, diagnostic location, existence prognosis prediction, by stages or imaging method may further comprise the steps: cell is contacted with compositions; (b) measure and the bonded radioactivity of cell; Therefore (c) shows tumor.

The example of suitable preparation comprises fluorescent dye, as FITC, PE etc., and fluorescin, as green fluorescent protein.Other example comprises Geigers and produces the enzyme that can discern variation (as change color) with substrate reactions.

In one embodiment, the preparation of test kit is a fluorescent dye, and as FITC, test kit provides the treatment of cancer effect Analysis, and the hemopoietic associated cancer of saying so more specifically is as leukemia, lymphoma and myeloma.Facs analysis is used to measure the percentage ratio and the staining power of the preparation staining cell of each phase of disease, during as diagnosis, during the treatment, catabasis and recurrence phase.

Antibody of the present invention and fragment thereof can with anticarcinogen, antineoplastic agent, antiviral agents, antimetastatic agents, anti-inflammatory agent, antithrombotic drug, anti-restenosis, anti-assemble medicine, anti-autoimmune medicine, anti-adhesive medicine, cardiovascular disease resistant medicine, medical substance or other disease-resistant medicine in conjunction with, is connected or otherwise association.Medicine refers to be used for mammal is carried out the medicine of prophylactic treatment or diagnosis, and described mammal is including but not limited to people, cattle, horse, pig, mice, dog, cat or any other homoiothermic animal.

The example of described medicine is including but not limited to the antiviral agents that comprises acyclovir, ganciclovir and zidovudine; Antithrombotic formation/the restenosis that comprises cilostazol, dalteparin sodium, Reviparin Sodium and aspirin; Comprise zaltoprofen, pranoprofen, drogelor, aspirin 17, diclofenac, ibuprofen, dexibuprofen, sulindac, naproxen, ammonia tolmetin, match the anti-inflammatory agent of examining former times, indomethacin, rofecoxib and nimesulide; The anti-autoimmune medicine that comprises leflunomide, denileukin diftitox, subreum, WinRho SDF, defibrotide and cyclophosphamide; And comprise limaprost, cloricromen and hyaluronic anti-adhesive/anti-medicine of assembling.

Exemplary medical substance comprises anthracycline antibiotics, as doxorubicin (amycin), daunorubicin (daunomycin), idarubicin, detorubicin, carminomycin, epirubicin, esorubicin, morpholine and substitutive derivative and compositions thereof.How exemplary medical substance comprises cisplatin, taxol, calicheamicin, vincristine, cytosine arabinoside (Ara-C), cyclophosphamide, prednisone, fludarabine, idarubicin, chlorambucil, interferon-ALPHA, hydroxyurea, temozolomide, Thalidomide and bleomycin, and their derivant, compositions and trim.

The inhibition of growth of cancer cells comprises, for example, (i) stop carcinous or the transitivity growth, (ii) slow down carcinous or the transitivity growth, the growth course or the transfer process that (iii) stop cancerous cell fully, and keep the survival of cell complete sum, (iv) contacting of interfere with cancer cells and microenvironment, or (v) kill cancer cell.

The inhibition of leukaemia's growth comprises, for example, (i) stop the growth of leukemia or transitivity, (ii) slow down the growth of leukemia or transitivity, the growth course or the transfer process that (iii) stop the leukaemia fully, and keep the survival of cell complete sum, (iv) contacting of interfere with cancer cells and microenvironment, or (v) kill the leukaemia.

Be used to connect antibody of the present invention and segmental disease-resistant medicine thereof, example anticancer and antileukemia comprises toxin, radiosiotope and medicine.

The example of toxin comprises gelonin, Pseudomonas exotoxin (PE), PE40, PE38, diphtheria toxin, diphtherotoxin, Ricin or their derivant, compositions and trim.

Radioisotopic example comprises and can be used for γ emitting substance, positron emission thing and the X ray emitting substance of locating and/or treating and β emitting substance that can be used for treating and α emitting substance.Aforementioned diagnosis, predict prognosis and the radiosiotope by stages of being used as also can be used for treatment.

Non-limiting anticancer or antileukemia example comprises anthracycline such as doxorubicin (amycin), daunorubicin (daunomycin), idarubicin, detorubicin, carminomycin, epirubicin, esorubicin and their morpholino and substitutive derivative, compositions and trim.Exemplary medical substance comprises cisplatin, taxol, calicheamicin, vincristine, cytosine arabinoside (Ara-C), cyclophosphamide, prednisone, daunorubicin, idarubicin, fludarabine, chlorambucil, interferon-ALPHA, hydroxyurea, temozolomide, Thalidomide, bleomycin and their derivant, compositions and trim.Preferred anticarcinogen or antileukemia are doxorubicin, morpholino doxorubicin or morpholino daunorubicin.

In one embodiment, the invention provides by giving the method for antibody induction of the present invention or activation ADCC.Accordingly, these antibody can activate ADCC and/or stimulate natural killer cell (NK) (as CD56+), T cytotoxic cell (as CD8+), and/or mononuclear cell, and they can cause lysis.In general, comprise the antibody or antibody moiety in Fc district after, described antibody combines with Fc receptor (FcR) on effector lymphocyte's (for example NK cell), causes the release of perforin and Cytotoxic cell proteinase-1, causes apoptosis.Various factors can influence ADCC, comprises the type, cytokine (for example IL-2 and G-CSF), incubation time, the quantity that is present in cell surface receptor and the antibody affinity that relate to the effector lymphocyte.

In one embodiment, Pharmaceutical composition of the present invention comprises antibody or its fragment and the pharmaceutical acceptable carrier of the scFv antibody fragment binding ability with SEQ ID NO:1.This antibody or its segmental amount are effective dose, described effective dose can effectively suppress or treat cell rolling, inflammation, infection, autoimmune disease, transfer, tumor cell or leukaemia's growth and/or propagation, or effectively suppresses or increase that leukaemia's quantity among tumor cell among the patient of tumor or the leukemic patient is suffered from treatment.Perhaps, antibody or its segmental amount are effective dose, can effectively increase tumor cell or leukaemia's mortality rate.Also or, antibody or its segmental amount are effective dose, can effectively change disease cell enantiopathy medicine damage, tumor cell to anticarcinogen damage or the leukaemia sensitivity to the antileukemia damage.Again on the one hand, antibody or its segmental amount are effective dose, can effectively reduce the quantity of leukaemia among the quantity of tumor cell among the patient who suffers from tumor or the leukemic patient.

Antibody of the present invention, construction, junctional complex and fragment can be passed through the patient that suitable method afford needs.Illustrative methods comprises in intravenous, intramuscular, subcutaneous, local, the trachea, in the sheath, in the intraperitoneal, lymph, administration in nose, Sublingual, oral, rectum, vagina, respiratory tract, oral cavity, intradermal, transdermal or the pleura.

For intravenous administration, it is the effective dose of the about 1000mg of about 0.1mg-that the preferred for preparation preparation makes the desired composition dosage that gives the patient.The dosage that more preferably gives desired composition is in the scope of the about 500mg of about 1mg-.The present composition is effective in bigger dosage range, and rely on many factors, such as the treatment disease concrete condition, based on the Pharmaceutical composition of peptide or polypeptide in the intravital half-life of patient, with medication, institute's treatment of the physics of antibody or its fragment and the compound medicine of Pharmaceutical composition and chemical property, Pharmaceutical composition or diagnose patient's concrete condition and treat the doctor and think important other parameter.

The Pharmaceutical composition that is used for oral administration can be any suitable form.Example comprises tablet, liquid agent, Emulsion, suspending agent, syrup, pill, capsule-type tablet and capsule.The method for preparing Pharmaceutical composition is well known in the artly (to see as Remington The Scienceand Practice of Pharmacy, Alfonso R.Gennaro (Ed.) Lippincott, Williams﹠amp; Wilkins (pub)).

Also can prepare Pharmaceutical composition of the present invention, with help regularly, continue, pulse or discharge continuously.Pharmaceutical composition of the present invention also can pass through the device administration, as regularly, continue, pulse or releasing device continuously.The Pharmaceutical composition that is used for topical can be any suitable form, as cream, ointment, lotion, patch, solution, suspending agent, lyophilized preparation and gel.Have antibody of the present invention, construction, junctional complex and segmental compositions and can comprise ethnopharmacology acceptable diluent, excipient, carrier etc.Tablet, pill, capsule-type tablet and capsule can comprise traditional excipient such as lactose, starch and magnesium stearate.Suppository can comprise excipient such as wax and glycerol.Injectable solution comprises aseptic apyrogeneity medium, as saline, also can comprise buffer agent, stabilizing agent or antiseptic.Also can use traditional casing.

Antibody of the present invention or its fragment and Pharmaceutical composition thereof can be used for treating the method (can comprise alleviation disease symptoms, prevent disease or suppress progression of disease as treatment) that needs patient disease.Described method comprises and suppressing or treatment cell rolling, inflammation, autoimmune disease, transfer, tumor cell or leukaemia's growth and/or propagation, or suppresses or quantity that leukaemia among tumor cell among the patient of tumor or the leukemic patient is suffered from treatment increases.In addition, described method also comprises the mortality rate that increases tumor cell or leukaemia, or changes the damage of disease cell enantiopathy medicine, tumor cell to anticarcinogen damage or the leukaemia sensitivity to the anticarcinogen damage.These class methods also comprise the quantity of leukaemia among the quantity of tumor cell among the patient who reduce to suffer from tumor or the leukemic patient.

The present invention also provides the method for eliminating the patient tumors cell, by the sample that contains patient's cell is provided, and with patient's cell and antibody incubation of the present invention.In one embodiment, the elimination tumor is carried out under exsomatizing.

Embodiment

List the following example and be used for helping to understand the present invention and further specify the present invention, but also should not be construed as restriction any scope of the present invention unintentionally.Though described special reagent and reaction condition, can it is made amendment and still belongs to category of the present invention.

Embodiment 1

Present embodiment has shown screening, production and the principal character of L32 scFv antibody fragment.Briefly, the phage display library of showing the scFv antibody fragment is used for obtaining and the productive target molecule, flow cytometry especially fluorescence-activated cell sorting (FACS) is used to identify and separates special phage clone, peptide or polypeptide recognition objective cell that described clone shows.Phage display library used herein makes up the peripheral blood lymphocyte from 49 non-immune people donors.

Select and identify phage clone by the multistep method that is known as biological elutriation.By phage display protein ligands variant (phage display library) and target cell incubation are carried out elutriation, remove not in conjunction with phage by washing technology, and the phage of specificity elution of bound.The optional amplification of the phage clone of eluting is the extra combination and the circulation of optional amplification then, and this helps comprising the enrichment that combines the segmental phage clone of optimum antibody with target.After the elutriation of number wheel, identify individual phage clone, by the corresponding dna sequencing of phage virus body is determined the peptide sequence that the clone shows.

In the present invention, the initial elutriation step of the screening of T lymphoma cell is carried out undefined epi-position, target cell (as B leukaemia, B-CLL cell, AML cell, multiple myeloma cells and metastatic cell) to needs carries out Immune Clone Selection subsequently, and described target cell surface markers is unknown.

Use the L1 scheme, find L32 scFv antibody cloning by elutriation phage display library on complete T lymphoma cell.This scheme starts from pre-wash.Be stored in-70 ℃, contain 2 * 10 ⁷The 1ml sample of individual freezing patient's leukemia/lymphoma T cell melts under 37 ℃ fast, is diluted in immediately in the cold 2%PBS-Lac Bovis seu Bubali of 10ml (MPBS).Cell descended 120 * g centrifugal 5 minutes in room temperature (RT), and washed twice is suspended among the MPBS, counts with hematimeter.Under the MRC agreement, use ScFv to show phage library (Nissim et al., EMBOJ., 13:692-98 (1994)).The library is configured at first shows the segmental phasmid of scFv library, wherein V _HAnd V _LThe territory is connected by flexible polypeptide.The N end of scFv that shows in the phasmid library and phage time capsid protein pIII merges, and sub-clone is to the pHEN1 carrier subsequently.All antibody fragment is at first reset the V gene by the never immune human peripheral blood lymphocyte of PCR and is produced (referring to " natural all ").For making whole antibody fragment variations, to 49 clones' human V _HIntroduce the random nucleotide sequence of the heavy chain CDR3 of 4-12 residue length of coding in the genetic fragment storehouse.The VL fragment that merges among all clones produces about 10 for the IGLV3S1 kind is the single V of variation gene ⁸Individual clone's single jar of library (single pot library).

The final volume that is chosen in of L32 scFv antibody cloning is that 0.5ml contains 10 ⁶Individual T cell, 10 ¹¹Individual phasmid clonogenic unit (CFU) (Nissim library) and 10 ¹³Carry out in the MPBS solution of individual wild type M13 phage, 4 ℃ are descended slow jolting 1 hour.Cell washing is the same then, and usefulness MPBS suspension cell is also centrifugal at 4 ℃ of following 120 * g.Selection and cell washing process repeat 3 times.

After the first round selects, by with cell at room temperature with the 150 μ l 0.1M glycine incubations of pH2.2 5 minutes, from T lymphoma cell elution of bound phasmid.After the neutralization, centrifugal and discard cell, collect and contain the particulate supernatant of wash-out bacteriophage, called after E1 stock solution.By adding 1ml TG-1 exponential phase of growth cell and in 37 ℃ of following incubations 30 minutes, this E1 storehouse of increasing.Portion places and is used for titration on the flat board, and the sample of all the other volumes places on the massive plate (150mm) that contains 2xTY/AMP (1.6% tryptone, 1% yeast extract, 0.5%NaCl and 100 μ g/ml ampicillin).Dull and stereotyped 30 ℃ of following incubated overnight.For determining every output of taking turns after the elutriation, the clone on the counting titration flat board, and calculate total output.

By cultured cell to A ₆₀₀Be 0.5-0.9 (exponential phase of growth), preparation is used for infecting the TG-1 bacterial strain and the fresh bacterial cultures of HB2151 bacterial strain of (amplification).Escherichia coli TG-1 cell is used to breed phage, and escherichia coli HB2151 cell is used to produce scFv albumen.Scrape and get and compile clone from massive plate.A (～10 ⁷) the amicillin resistance escherichia coli TG-1 cell of liquid culture grows to A ₆₀₀For about 0.5, (VSC-M13 Stratagene) infects the phasmid storehouses that produce a large amount of amplifications to use helper phage then.Phasmid reclaims (Harrison et al., Methods in Enzymology (1996) 267:83-109) with the PEG intermediate processing.About 10 ¹²The amplification E1 stock solution of phasmid/ml such as the above-mentioned elutriation that is used for next round with the T cell.

Second carries out with the basic method identical with first round elutriation with third round " order elutriation ", only does following change: (i) second take turns elutriation, use about 10 ¹¹Individual phasmid is after (ii) selecting and washing, by cell at room temperature being used PBS/1%BSA+ATP (10mM) incubation 15 minutes, elution of bound phasmid.Centrifuge cell is also collected supernatant.Comprise this method second and take turns the product called after E1AT1 of eluting phasmid, described product without amplification as above-mentioned be used for the order elutriation third round.Take turns as second, behind the ATP eluting, phasmid such as above-mentionedly in the TG-1 cell, increase.Final amplification stock solution called after E1AT2.A (5 μ l) is mixed for infection, titration and sequence analysis with the TG-1 culture.Residual volume (45 μ l) is used for amplification and preservation with 1.3ml TG-1 Cell sap incubation.

In three consecutive steps of the biological elutriation scheme of L1, that estimates to be used for the phasmid quantity (inputs) of elutriation and estimation eluting is summarized in table 2 in conjunction with phasmid quantity (output).List the cell source in the table and obtained the eluting medium of every kind of product, and the condition that is used to offer an explanation each independent storehouse.

Table 2

The stock solution that drops into	The cell source	Eluent	Output	The stock solution of amplification
The stock solution that drops into	The cell source	Eluent	Output	The stock solution of amplification	Nissim library-10 ¹²????E1-1011 ????E1AT1-500	T lymphoma T lymphoma T lymphoma	Acid ATP (10mM) ATP (10mM)	????3.3×10 ⁶????500 ????37	??E1 ??E1AT1 ^*??E1AT2

^*Eluting but not amplification.

Result in the table 1 shows, when the ATP eluent is used for second and during third round, the quantity of wash-out bacteriophage seldom shows that the phage specificity may increase.

After the elutriation, the every wheel selects some clone to be used for order-checking.The aminoacid sequence that this part provides is as follows: 1 time sequence (a) occurs surpassing in selected clone, (b) the only sequence in heavy chain CDR3 district (VH-CDR3) and (c) kind of every kind of separating clone VH kind system.

Table 3

The clone	The VH-CDR3 size	The VH-CDR3 sequence	Plant system	Product	Frequency
The clone	The VH-CDR3 size	The VH-CDR3 sequence	Plant system	Product	Frequency	? ??L32 ? ? ? ??L31	? ????8 ? ? ? ?????7	Leu?Asn?Pro?Lys?Val?Lys?His ????????????Met ? ????(SEQ?ID?NO：4) Leu?Arg?Gly?Gly?Asn?Ala?Met ? ????(SEQ?ID?NO：5)	? ???VH3- ???DP32 ? ???VH3- ? ???DP32	? ??E1AT ??2 ? ??E1AT ? ??2	? ? ??4/15 ? ? ??5/15

Identified 2 classes clone after the biological elutriation scheme of L1, L32 and L31, their sequence sees Table 3.The quantity (VH-CDR3 size) and the specific C DR3 sequence that have shown amino acid residue in the CDR3 district in the table, and the name of every kind of clone's kind system.In addition, shown the isolating number of times of special clone's type, always checked order as the L1 scheme and clone the function (frequency) of quantity.What is interesting is that though the library comprises the VH in 5 different VH families (VH1, VH2, VH3, VH4 and VH6) source, wherein VH3 accounts for and utilizes about 47%, the 2 kind of isolating clone of gene to belong to VH3 family (DP32).This is favourable for the scFv purification process, and this is because the protein A agarose is used for the scFv that purification derives from VH3 family, and can not be used for the clone that purification derives from family beyond the VH3.

Embodiment 2

Present embodiment has been described the production of using the scFv antibody that compares in the various comparative studies.

U. S. application number 10/032,423; 10/032,037; 10/029,988; 10/029,926; 09/751,181 and 60/258,948 and international application no PCT/US01/49442 and PCT/US01/49440 in describe separating and sign of Y1 and Y17 scFv antibody cloning in detail.

In addition, selected negative control scFv clone.In conjunction with experiment, (before selecting) selects monospecific polyclonal from natural library for all.From this clone's preparation phage stock solution and solubility scFv, called after N14.Sequence analysis shows that it belongs to the VH4-DP65 gene family.The 11-mer V of this clones coding _H-CDR3 sequence called after N14 CDR3 is SEQ ID NO:6.Another kind of negative clone N01 is used for the binding analysis experiment.N01 clone (with the reaction of recombinant hepatitis B virus [HBV] granule) belongs to V _H3-DP35 family, the 9-mer V of this clones coding _H-CDR3 sequence called after N01 CDR3 is SEQ ID NO:7.

By elutriation phage display library on T lymphoma cell film, separate TM scFv antibody cloning (following) with the TM scheme.In this scheme, described in top L1 scheme, pre-wash T cell (2 * 10 ⁷).Behind the pre-wash T cell, contain 10 by adding 2ml ¹²The MPBS of individual phasmid (deriving from original Nissim library) selects on fixed T lymphoma cell film.The slow jolting of test tube 30 minutes, not jolting incubation 90 minutes (reaction of 2 steps is at room temperature carried out) then.Decant in vitro tolerantly, remove, with PBS and the washing of 0.1% tween 10 times, then with PBS washing 10 times not in conjunction with phasmid.For eluting, directly in test tube, add the escherichia coli TG-1 cell (2ml) of exponential growth, 37 ℃ of following incubations and slow jolting.Portion places and is used for titration on the flat board as mentioned above, and residual volume places and is used for amplification on the flat board.In addition, amplification is carried out as described in the L1 scheme.2 take turns middle option program repetition 2 times in addition, use 10 ¹¹The phasmid of individual aforementioned amplification stock solution.Fixedly clone called after TM1.1-myc+/TM1.1 for first kind of the amplification of the third round elutriation program on T cell membrane stock solution.Variant (TM1.1-myc+) from this clone some scFv antibody, especially TM1.1 of preparation and band myc labelling thereof.

Except the TM1.1-myc+ clone, separate following other clone with the TM scheme.The amplification bank of third round film elutriation is used for the complete T lymphoma cell of elutriation.This method is undertaken by top L1 scheme is described basically, uses 2 * 10 ⁷Individual cell and 10 ¹⁰Individual phasmid.4 ℃ of down insulations after 2 hours with 50 μ l trypsin: EDTA (0.25%:0.05%) elution of bound phasmid from the washed cell mass, add 50 μ l FCS then and neutralize.For titration and amplification, use 1ml escherichia coli TG-1 culture (A ₆₀₀=0.5).Amplification stock solution called after TM2 is used for another and takes turns elutriation on the T lymphoma cell, as mentioned above.Final stock solution called after TM3.According to the TM scheme, the sequence of the scFv that is separated to is as shown in table 4.Estimated that also the combination behind the ScFv FITC labelling is active, to confirm the specific reservation of scFv (referring to embodiment 7).For example, TM3.13 combines by it according to facs analysis with the bonded specificity of T-ALL cell and is proved (referring to accompanying drawing 1).

Table 4

The clone	The VH-CDR3 size	The VH-CDR3 sequence	Plant system	Product	Frequency
The clone	The VH-CDR3 size	The VH-CDR3 sequence	Plant system	Product	Frequency	??TM2.3 ??1 ??TM2.2 ??3 ??TM3.2 ??0 ??TM3.1 ??8 ??TM3.1 ??3	????7 ? ????7 ? ??7 ????7 ????11	Leu?Thr?His?Arg?Ser?Ser?Arg Thr?Gln?Arg?Arg?Asp?Leu?Gly Lys?Arg?Val?Ser?Leu?Leu?Thr Ser?Tyr?Arg?Arg?His?Ser?Arg Arg?Asp?Lys?Thr?Thr?Asn?Phe?Tyr Phe?Met?Lys	?VH3- ?DP46 ?VH3- ?DP53 ?VH3- ?DP70 ?VH3- ?DP47 ?VH3- ?DP26	TM2 TM2 TM3 TM3 TM3	?2/10 ?5/10 ?1/7 ?2/7 ?1/7

Embodiment 3

Present embodiment has shown L32 scFv clone's production, purification, labelling and sign.

For the production of solvable scFv, the carrier pHEN1 that is used to make up original phasmid library is designed to have a succinum termination codon at scFv and pIII gene junction.Therefore, when infecting by phasmid, when selected clone's carrier was introduced the escherichia coli HB2151 of unrestraint gene, this system can produce solvable scFv and it is secreted into (Harrison et al., Methods in Enzymol.267:83-109 (1996)) in the antibacterial kytoplasm.ScFv can easily reclaim from culture medium then.Solvable scFv induces production (Gilbert and Muller-Hill, PNAS (US) 58:2415 (1967)) with IPTG (isopropylthiogalactoside) under the regulation and control of lacZ promoter.

The sequence (SEQ ID NO:8) of 10 aminoacid c-myc labellings of encoding is included in the upstream that succinum changes in the carrier.The C end of expressing scFv should carry the c-myc labelling, and described labelling can detect (deriving from the 9E10 hybridoma at European cell culture preservation center (ECACC)) with mouse anti myc traget antibody.

The scFv of selected clone and contrast clone N01 belongs to VH3 family, can be on protein A affinity post purification.Preparation is incubated with protein A sepharose 4B (Sepharose beads) from the pericentral siphon part (100-250ml) of every kind of clone's inducing culture thing.Take off with pickling that (the 0.1M glycine pH3.0) reclaims in conjunction with scFv, uses among the Tris pH8.0 subsequently and eluent.Reclaim proteic concentration by measuring A ₂₈₀Determine, subsequently by dialysing or on the G-25 agarose column, carrying out the PBS buffer exchange.

The scFv that derives from L1 scheme clone L32 also belongs to VH3 gene family (DP-32).But, after extracting from pericentral siphon, need before sample pipetting volume is to the protein A affinity post, add 5mM DTT, then as above-mentioned purification and recovery from the protein A sepharose 4B, carry out the PBS buffer exchange.

The scFv of negative clone N14 belongs to the VH4 gene family, can not be on protein A affinity post purification, therefore purification on Sephacryl S-200 post.By with 60% ammonium sulfate precipitation 200ml inducing culture thing pericentral siphon part total protein, purification scFv N14.Precipitation is suspended in 2ml0.1 * PBS, and 5mM EDTA, 5mM PMSF, application of sample arrive the Sephacryl S-200 post of usefulness running buffer (0.1 * PBS, 5mM EDTA) pre-equilibration (on 1.5 * 90cm).Fraction collection albumen merges the part (detecting with SDS-PAGE and western blot analysis) that contains N14 scFv, and lyophilization is suspended in the water of 1/10 volume.N14 scFv (unmarked and FITC labelling) is used as negative control in the facs analysis experiment then.

The scFv of purification uses the FITC labelling then.The purification scFv of the about 1mg of each specimen is suspended in PBS, and by manufacturer explanation, (Sigma-Aldrich Corp., St.Louis MO) make itself and FITC coupling to use fluorescent labeling FITC coupling commercial reagents box.Behind purification and FITC labelling, with the HPLC (A of SDS-PAGE, Western blotting, use Superdex-75 post ₂₈₀And A ₄₉₅) and fluoremetry analyze the feature of each specimen (labelling with unlabelled).The purity that the analysis showed that N14 scFv is 80%, and VH3 clone's purity is 90%, and each scFv molecule combines (the F/P ratio is 2: 1) with about 2 FITC molecules.

Embodiment 4

Present embodiment has shown combining of L32 scFv and washing platelet and platelet rich plasma (PRP), and L32 scFv is to the effect of platelet aggregation.

For platelet aggregation research, collect blood with the test tube that contains 3.8% sodium citrate.By centrifugal 10 minutes preparation PRP under 250 * g.Platelet concentrate from blood bank's acquisition acid-citrate-dextrose (ACD).Separate platelet, wash once, and wash with 1: 7 ratio with saline with the buffer that contains ACD.Each washing back platelet under 800g centrifugal 10 minutes, and be suspended in Tyrodes solution (2mM MgCl ₂, 137mM NaCl, 2.68mMKCl, 3mM NaH ₂PO ₄, 0.1% glucose, 5mM Hepes and 0.35% albumin, pH7.35) in, counting cells quantity.

Lumiaggregometer (Chronolog, Havertown, PA) in, stir with 500rpm under 37 ℃ of PRP in the whole blood and the washing platelets.Light transmittance difference by platelet suspension and suspension media between them is considered to 100% gathering.Before adding agonist, estimate the effect of L32 by the L32 that adds variable concentrations to platelet aggregation, write down result's (accompanying drawing 2) of 4 minutes.

Assemble mensuration with washing platelet.Reactant mixture contains 2 * 10 ⁸The pig von Willebrand factor of washing platelet/ml and 4 μ g/ml.37 ℃ of insulations added the ristocetin (gathering agonist) of 0.4mg/ml after 3 minutes, write down 4 minutes aggreation.L32 and other scFv antibody are estimated by add the described scFv of 50 μ g/ml before adding agonist the effect of platelet aggregation, write down 4 minutes result.(N=2) test L32 relies on agglutinative effect to washing platelet vWF in 2 washing platelet samples, to agglutinative elimination, can be observed normal coagulation (90% coagulation) with respect to the scFv Y1 of known effect.The effect of the isolating TM1.1-myc+ of evaluation employing TM scheme in contrast.Under the same conditions, Y1 scFv antibody suppresses about 70% the inductive platelet aggregation of ristocetin (accompanying drawing 2).

Measure for the gathering of using PRP, reactant mixture contains PRP (about 2 * 10 ⁸/ ml).Behind 37 ℃ of insulation 3 molecules, add lmg/ml ristocetin (gathering agonist), write down 4 minutes aggreation.Light transmittance difference by platelet suspension and PPP (platelet poor plasma) between them is considered to 100% gathering.By add 50 μ l/mlL32 scFv before adding agonist, evaluation to the effect of platelet aggregation, is write down 4 minutes result from L32 among 3 different donors' (N=3) the PRP.Can be observed normal gathering (90% assembles) (accompanying drawing 2) after in PRP, adding L32 scFv.As mentioned above, the effect that compares L32 and TM1.1 and Y1.Similar to washing platelet, Y1 suppresses about 70% PRP platelet aggregation.

Can reach a conclusion, the clone L32 of 50 μ g/ml does not have remarkable inhibition effect to the platelet aggregation of washing platelet or PRP.

Hematoblastic L32 scFv dyeing (combination) is also estimated by FACS.This method is used for the mensuration based on the fluorescent labeling staining power.As shown in Figure 3, obtain the dyeing blood platelet with Y1 and Y1-myc+scFv (the sugar small cup albumino reaction Y1 scFv antibody that has the c-myc labelling) dyeing from PRP.On the contrary, and compare, dye after the described platelet with L32 scFv with control antibodies dyeing, relatively change of fluorescence signal (rectangular histogram in the comparative drawings figs 3, wherein TM1.1-myc+ for not with the bonded scFv of any platelet associated epitope).

Embodiment 5

The combination facs analysis of scFv L32 and various different cell lines.(i) L32 is being used in analysis; (ii) anti-single-chain antibody; Carry out after the (iii) anti-rabbit FITC traget antibody 3 step dyeing.According to dividing different cell lines in conjunction with back cell colony geometrical mean with the ratio of negative control, as shown in table 5 with L32.Low combination refers to that ratio is 1 cell, and medium combination refers to that ratio is the cell of 1-4, and is high in conjunction with referring to the cell of ratio greater than 4.

Table 5

The L32 height	Among the L32	L32 is low
The L32 height	Among the L32	L32 is low	????KG-1 ????Jurkat	????Molt-4 ????Hut78 ????HEL ????K562 ????CCRF-CEM ????HL60	????Raji ????Daudi ????UMUC3 ????Namalwa

Investigate between Y1 and the L32 competition by described single-chain antibody to same binding site on these cells.In a this experiment, estimate unmarked antibody and biotin labeling Y1 (Y1-myc+) competition to KG-1 cell (deriving from AML patient's human cell system).The result is shown in attached Figure 4 and 5.As shown in the figure, L32 competition Y1 combines with the KG-1 cell, is similar to Y1 scFv itself.This result is supported in preliminary radio receptor assay research, and L32 scFv dose dependent is partly replaced in the described research ¹²⁵The Y1 scFv of I labelling and combining of KG-1 cell.

In competition research, estimate unmarked antibody combines the KG-1 cell with biotin labeling L32 scFv (0.5,2 or 5 μ g) competition ability.U. S. application number 10/032,423; 10/032,037; 10/029,988; 10/029,926; 09/751,181 and 60/258,948 and international application no PCT/US01/49442 and PCT/US01/49440 in the result show that Y1 and anti-CD162 antibody competition combine with CD162 is antigenic.Facs analysis is used for the measurement markers antibodies, and the result is expressed as bonded geometrical mean.The described table 6 that the results are summarized in shows Y1 scFv and the metathetical concentration dependent of anti-CD162 antibody.When the amount of unmarked antibody is far longer than the amount of traget antibody, replaced by specific antibody above 70% combination, combination has no significant effect but not specificity T M1.1scFv is to L32.Though anti-CD162 has the ability (up to 89%) of the strongest displacement L32, Y1 and L32 scFv antibody are to replace the combination of L32 in various degree.Based on described and other result, compare only less L32 and L32 and the Y1 competition of need with Y1, show that same loci L32 is had stronger binding ability than Y1.Therefore, these results have further supported the bonded specificity of L32, and the fundamental relation between L32 epi-position and Y1 and the anti-CD162 antibody identification meter position.

Table 6

Inhibitor concentration	The L32 biotin concentration
	The L32 biotin concentration			??0.5μg?L32-B	??2μg?L32-B	??5μg?L32-B
	There is not the anti-CD162 of 50 μ g TM1.1,50 μ g Y1 50 μ g L32,5 μ g	????22.65 ????21.6 ????6.36 ????5.66 ????4.06	????28.3 ????12.99 ????9.34 ????3.95	??0.5μg?L32-B	??2μg?L32-B	??5μg?L32-B	????38.32 ????21.99 ????22.58 ????4.4

Further estimate competition between L32, Y1 and the anti-CD162 antibody by measuring anti-CD162 antibody of labelling and the bonded displacement degree of KG-1 cell.Find that L32 and Y1 scFv antibody (50 μ g) all can make the geometrical mean of anti-CD162 (5 μ g) labelling KG-1 cell reduce about 82%.Therefore, the epi-position of described antibody obviously is closely related.

Embodiment 6

Present embodiment confirms combining of L32 scFv antibody and GPIb proteolytic fragments sugar albumen (GC) by ELISA.

As Michalson (as described in the Blood 67:19-26 (1986), from Freshman platelet purification GC.Before being used for mensuration, determine GPIb by the EIA method with 2 kinds of commercial monoclonal antibody formulations of difference; First kind of antibody (H1P1 clone), available from Phannigen (San Diego CA), suppresses the inductive platelet aggregation of ristocetin, second kind of antibody preparation (PM6/40 clone), available from Serotec Inc. (Raleigh, NC), anticoagulant not.

By combining of ELISA quantitative analysis L32 and GC.By 4 ℃ of following incubated overnight, wrap by the Maxisorp plate hole with the GC that is diluted to 1 μ g/ml (PBS * 1).After removing excessive GC, plate at room temperature sealed 1 hour with PBSTM (phosphate buffered saline(PBS) that contains 2% Lac Bovis seu Bubali and 0.05% tween).After the thorough washing of PBST, plate and the scFv insulation that is diluted to various concentration with PBSTM.Subsequently, the anti-myc insulation of the anti-scFv of plate and 1: 250 dilution or the anti-VL of dilution in 1: 50 or dilution in 1: 100, subsequently with PBSTM 1: 25, the anti-rabbit HRP (Jackson) of 000 dilution or 1: 25, the anti-mice HRP insulation (associated antibodies) of 000 dilution.Carry out chromogenic reaction with TMB, add 0.5M sulphuric acid stopped reaction.Read plate with the ELISA reader under 450nm, the result of average double sample calculates the ELISA unit value by subtracting background from meansigma methods (no first antibody dyeing) OD value.The results are shown in accompanying drawing 6, show GC and Y1 binding ratio L32 combine about 3-4 doubly.

Embodiment 7

Present embodiment shows L32 scFv antibody and various people proteic combination of originating by western blot analysis.

Preparation cell extract (lysate).Collecting cell (2 * 10 ⁶), centrifugal in microcentrifugal tube (1300rpm, 4 ℃, 5 minutes).With 0.5-1ml PBS washing precipitation, soft mixing, mixture such as preceding centrifugal.Repeat the washing with 0.5-1ml PBS, agglomerating cell is with lysis buffer (200 μ l/20 * 10 that suspend ⁶Cell mass).Though can use other suitable lysis buffer, used lysis buffer is 50mM Tris pH7.4,1mm PMSF, 1%NP-40 and 1mM EDTA.Suspension is incubated about 60 minutes on ice, centrifugal then (3000rpm, 4 ℃, 5 minutes).Collect supernatant then, be divided into several parts.

Also prepare thick film fragment and memebrane protein extract.The homogenize buffer of 20 times of volumes joins in the packed cell of 1 times of volume.The homogenize buffer contains 2% (w/v) polysorbas20,1mMMgSO ₄, 2mM CaCl ₂, 150mM NaCl and 25mM Tris-HCI, pH7.4.Add following protease inhibitor: 1mM PMSF, 5 μ g/ml Leupeptin and 5 μ g/mlAprotonin.Potter-Elvehjem homogenizer (Ultra-Torex) homogenate that cell rotates the Teflon mallet with band, speed is 3-5 time.Sample keeps cooling when homogenate, stirred 1 hour in the ice bath then.Sample grinds in homogenizer for several times again, and 4 ℃ of following 3000g centrifugal 30 minutes then.Collect supernatant, centrifugal 1 hour of 4 ℃ of following 45000g (ss-34 rotor 19000rpm).Discard the centrifugal supernatant of 45000g.Add the solution that contains 50mM Tris 7.4,1mM EDTA, 1%NP-40 and protease inhibitor to precipitation, dissolved precipitation placed 1 hour on ice.

Blood bank's healthy individual blood plasma that will compile is diluted to 1: 10 after (v/v) with PBS, preparation plasma protein sample.Solution after the dilution is through 0.45 μ m membrane filtration, with several parts of stored frozen (20 ℃) until analysis.Sample is compressed into capable 10%SDS-PAGE with the 140-160 voltaism at Sigma Z37 on the 503-9 electrophresis apparatus then, and the time is about 3.5 hours.(20%MeOH, 192mM glycine, 25mM Tris pH8.3) with 20 volts of voltage ambient temperature overnight, are transferred on the nitrocellulose filter sample behind the electrophoresis in the Tris glycine buffer.

Nitrocellulose filter is with sealing 1 hour under the 5% skimmed milk room temperature.Film is then at room temperature with 0.05% polysorbas20 washing 3 times, each 5 minutes.Then film under room temperature with 2% skimmed milk and PBS in the 5 μ g/ml Y1-biotin, L32-biotin that contain or the insulation of TM1.1-biotin 1 hour.(about 10 ℃ of about 4-) washs film 3 times, each 5 minutes with the cold PBS that contains 0.05% polysorbas20 in the cold house then.Film and the SAV-HRP that contains dilution in 2% skimmed milk and 0.05% tween 1: 1000 (strepto-affinity element-HRP) (ultimate density is 1 μ g/ml) insulation in the cold house then.(about 25 ℃) are at room temperature carried out in dilution, and the SAV-HRP of dilution was at cooled on ice 10-15 minute before using.Carried out about 1 hour under the soft vibration of insulation.

After the SAV-HRP insulation, as above-mentioned washing film.By the guidance of commercialization scheme, film and Super Signal mixture (Pierce) insulation 5 minutes, dry excess solution.Film is exposed to X-ray film (Fuji), makes film development.The result of these researchs (accompanying drawing 7) shows, the protein binding on L32 and the leukaemia, and described molecular weight of albumen is the molecular weight of PSGL-1, about 105kD.In addition, L32 and Y1 and identical GC band and KG-1 cell effect.But L32 combines well below Y1 with plasma protein, and should be negative greater than being with of 100kDa in the Raji cell extract.

Embodiment 8

Present embodiment shows combining of L32 and Y1 and sulphation and non-sulfuric acid peptide comparability by ELISA.

Preparation is combined to peptide based on the sulphation and the non-sulfuric acid of suitable epi-position (the 268-285 amino acids of GPIb and the 1-17 amino acids of ripe PSGL-1), is used to estimate L32 scFv antibody binding specificity (ELISA).Sulphation and non-sulfuric acid peptide (1 μ M) are connected in the microtitration plate of suitable elisa assay, the peptide that thorough flush away does not connect before nonspecific binding site is closed.Plate at room temperature with shown in concentration scFv antibody (referring to accompanying drawing 8) insulation 1 hour.The scFv that connects is with the anti-V of multi-clone rabbit _LAntibody test is the anti-rabbit anti-serum of horseradish peroxidase (HRP) subsequently.Sample stops peroxidase reaction with 0.5M sulphuric acid with substrate TMB colour developing after about 10 minutes.Relatively L32 and these peptides combines and combine (as the negative control) of Y1 and TM1.1 scFv antibody and peptide.Subtracting background dyeing (during no first antibody) from the value of each acquisition obtains the value that bar diagram shows.Be used to estimate bonded peptide and basic structure character is shown in table 7.

Table 7

The source of peptide	Name	Sequence	?AA	?MW	Sulphation
The source of peptide	Name	Sequence	?AA	?MW	Sulphation	The former PSGL-1 N end of the former γ of fibrinogen γ fibrillin PSGL-1 N end PSGL-1 N end PSGL-1 N end PSGL-1 N end PSGL-1 N end PSGL-1 N end PSGL-1 N end GPIb α GPIb α GPIb α GPIb α CCR5 CCR5	??A ? ??B ? ??C ??D ??E ? ??F ? ??G ??H ? ??I ??J ? ??K，P1 ? ??L，P1S ? ??P14S ? ??P28S ??M ??N	?VRPEHPAETEYESLYPED ?DL ?VRPEHPAETEY ^ESLY ^P ?EDDL ?QATEYEYLDYDFLPETE ?QATEY ^EYLDYDFLPETE ?QATEY ^EY ^LDYDFLPET ?E ?QATEY ^EYLDY ^DFLPET ?E ?QATEYEY ^LDYDFLPETE ?QATEYEY ^LDY ^DFLPET ?E ?QATEYEYLDY ^DFLPETE ?QATEY ^EY ^LDY ^DFLPE ?TE ?GDEGDTDLYDYYPEEDT ?E ?GDEGDTDLY ^DY ^Y ^PEE ?DTE ?GDEGDTDLYDYY ^PEED ?TE ?TDLY ^DYYPEEDTE ?MDYQVSSPIYDINYYTSE ?MDY ^QVSSPIY ^DINY ^*Y ?TSE	?20 ? ?20 ? ?17 ?17 ?17 ? ?17 ? ?17 ?17 ? ?17 ?17 ? ?18 ? ?18 ? ?18 ? ?13 ?19 ?18 ?	2389 ? 2549 ? 2126 2206 2286 ? 2286 ? 2286 2286 ? 2286 2286 ? 2126 ? 2366 ? 1732 ? 1732 2189 2429	-sulphation-sulphation sulphation sulphation sulphation sulphation sulphation sulphation-sulphation sulphation sulphation-sulphation

Y ^*Expression sulphation tyrosine

Obtained significant dosage correlation L32 scFv antibodies (accompanying drawing 8) with F, H, I and J peptide (i.e. Sulfated PSGL-1 related peptides on the 3rd tyrosine residue) and L and P28S peptide (i.e. sulphation GPIb α related peptides on first tyrosine residue).L32 scFv antibody is very similar with combining of I with combining with it of J.Though the combination of its relevant L with GPIb (P1S) peptide is a significance, this is lower than the result who obtains with the PSGL-1 related peptides.Y1 or L32 all do not find significantly to combine with sulphation PSGL-1 related peptides G and D (lacking sulphation at the 3rd residue).In addition, Y1 or L32 all not with the combination of the Fibrinogen γ relevant sulphation of chain (or non-sulfuric acidization) peptide.Do not have the not Sulfated peptide of scFv antibody and tyrosine to combine, show that sulphation is necessary for combination.In addition, the data of GPIb related peptides P28S show, the sulphation of first tyrosine is for being significance in conjunction with GPIb, and compare with E, and the data of PSGL-1 peptide I and J show, the sulphation of the 3rd tyrosine is for being significance in conjunction with PSGL-1.Though for sulphation peptide (in this measures), Y1 and L32 show basic identical pattern in conjunction with behavior, Y1 shows higher affinity than L32 under the various situations.

From above-mentioned experimental result and U. S. application number 10/032,423; 10/032,037; 10/029,988; 10/029,926; 09/751,181 and 60/258,948 and international application no PCT/US01/49442 and PCT/US01/49440 in data presented, can reach a conclusion, the epi-position of L32 scFv antibody wherein has negative charge aminoacid bunch between the 1-17 of ripe PSGL-1 amino acids.

PSGL-1 is that E, L and P select proteic receptor, identifies based on competitive assay as the part of Y1 antibody, and wherein the anti-PSGL-1 antibody of the different commercialization supplies with being combined in of KG-1 cell of Y1 antibody carries out under existing.The N petiolarea of PSGL-1 comprises sulphation tyrosine residue and negative charge aminoacid bunch.(U. S. application number 10/032,423; 10/032,037; 10/029,988; 10/029,926; 09/751,181 and 60/258,948 and international application no PCT/US01/49442 and PCT/US01/49440).

Though Y1 antibody combines with some molecule, as sugar small cup protein molecular, Fibrinogen γ ', human plasma complement chemical compound 4 and PSGL-1 molecule on the platelet, itself and the affinity ratio of the primary leukemia cell that derives from AML or multiple myeloma (MM) patient and the affinity height of aforementioned epi-position.

Do not wish to be subject to any particular theory, the affinity that Y1 is higher with the sulphation peptide (comparing) with L32, and L32 and the leukaemia system affinity (with Y1 compare) the same (as following embodiment 10,16 and 17 as shown in) higher with malignant cell, may be owing to the structure picture of relevant peptide sequence or the possible difference of exposure in the total length native protein on various factors such as the cell.For example, the peptide that is used to test is linear, secondary when not having its native state and tertiary structure.These differences are not obvious in non-limiting linear synthetic peptide.Therefore, the potential therapeutic use of L32 antibodies of cell system proposition may not observe in artificial peptide system.

Embodiment 9

U. S. application number 10/032,423; 10/032,037; 10/029,988; 10/029,926; 09/751,181 and 60/258,948 and international application no PCT/US01/49442 and PCT/US01/49440 in the result show that Y1 has similar recognition feature with Y17 antibody on platelet.Present embodiment shows the tyrosine sulphation of GPIb source peptide and sudden change to Y17 scFv antibody and the bonded influence of washing platelet, and Y17 and PSGL-1 the combining the Sulfated dependence of tyrosine of peptide of originating.

GPIb source peptide is selected from table 6, and other 2 have following sequence of N end and shorten GPIb source peptide and be used for these research: P2S-TDLY ^*DY ^*Y ^*PEEDTE and P25S-TDLYDY ^*Y ^*PEEDTE.

Measure combining of Y17 scFv antibody and washing platelet by FACS, as described in example 4 above.By add before the platelet transfusion at first with Y17 with shown in peptide (referring to the accompanying drawing 9) insulation of concentration, estimate various peptides to Y17 and the bonded influence of platelet.

All are tried in the peptide, and the GPIb source peptide (P28S) that comprises 276 sulphation tyrosine farthest suppresses Y17 and combines with washing platelet.Preparation comprises the GPIb source peptide that aminoacid changes, and the consensus sequence requirement of Y17 identification is confirmed in test.These results show that first sulphation tyrosine combines very important for Y17 with washing platelet.But the sulphation of second tyrosine is obviously inoperative in Y17 identification.277 and 275 aspartic acid negative charge amino acid residue (D) is also very important for the combination of Y17.This result is to similar with the observed result of Y1.

For confirming decision Y17 scFv antibody and the bonded condition of PSGL1, use with the bonded PSGL1 of plate source peptide and carry out elisa assay.What the Y17 scFv antibody of 5 μ g/ml and 20 μ g/ml was firm combines with two kinds of PSGL1 source peptides, promptly contains the I of sulphation tyrosine on the 3rd and comprises the J of 3 sulphation peptides.Accompanying drawing 10 shows, Y17 scFv and combining of J are a bit larger tham combining of Y1 and J, and the association class of two kinds of scFv and I seemingly.They are stronger with combining of J for they and the binding ratio of I.Y1 or Y17 all do not combine (not having Sulfated PSGL1 peptide) significantly with G.

In a word, in the peptide level, all 3 clones Y1, Y17 have similar specific characteristics with L32.

Embodiment 10

Present embodiment shows L32 and combining from normal volunteer's primary cell and leukaemic's primary cell.

All bacterial clones are cultivated, are induced, the method for scFv antibody fragment results and antibody fragment purification is according to Harrison et al., and ditto carry out (1996).Basically, any each scFv clone more than 2 kinds or 2 kinds can be selected from Nissim I antibody phage display libraries, and with preparation rabbit source polyclonal antibody, described antibody can be discerned the individual scFv antibody in any Nissim of being present in library or contain identical V _LOr its segmental any IgG or its fragment.

The polyclonal antibody antagonism that produces derives from the VL of the scFv antibody cloning (Y1) of Nissim I antibody phage display libraries.The dna fragmentation in coding people antibody VL territory is that (identical dna fragmentation can obtain (Nissim et al. from other any clone in Nissim I library to the PCR clone who clones from Y1, (1994), ditto) in addition same methods obtain from the people's gene group), use following synthetic oligonucleotide primer thing: oligo 5 '-NdeI (TTT CATATGGAGCTGACTCAGGACCCTGCT) and oligo 3 '-EcoRI (TTT GAATTCCTATTTTGCTTTTGCGGC).With (PCR condition: 94 ℃ 1 minute after the PCR amplification, 56 ℃ 2 minutes, 72 ℃ 2 minutes * 30 times, then 65 ℃ 5 minutes), the dna fragmentation that obtains NdeI and EcoRI digestion with restriction enzyme, and be cloned among the NdeI and EcoRI restriction endonuclease sites of predigestion plasmid, described plasmid is the IPTG inducible expression carrier that is used for the recombiant protein prokaryotic expression in the escherichia coli.

Bacillus coli cells is selected positive colony with above-mentioned oligonucleotide primers by pcr amplification with connecting the mixture transfection.Cultivation comprises the cell of this plasmid and uses the IPTG abduction delivering.After IPTG induced, the growth of 22 ℃ of 1L cultures was after 16 hours, centrifugal collection bacterial cell.Separate inclusion body, it is dissolved among guanidine-HCl+DTE, folding again by being diluted in the buffer that comprises Tris-arginine-EDTA.5-10 ℃ folding again after 48 hours, dialyses in 20mM glycine (pH9) and concentrate and contain proteic solution.Contain proteic solution after the dialysis with ion exchange column HiTrapQ purification again, with gradient NaCl eluting.Main peak SDS-PAGE and gel filtration analysis.From the 1L culture, obtain the V of 10mg purification at least _L

Rabbit is used V _L(400mg) and CFA (complete Freund's adjuvant) immunity, then with the interval V in 2-4 week _L(200mg) and IFA (incomplete Freund's adjuvant) immunity.The titre that obtains is lower by (1: 50-1: 100), may be owing to V between people and the rabbit _LHomology is higher.

Use directly from the anti-scFv antibody of polyclone rabbit anteserum after the immunity or behind purification on the protein A agarose column, detect scFv antibody with through the facs analysis cell combine or with isolating various proteic combinations of SDS-PAGE (western blot analysis).

In general, one of 3 kinds of facs analysis are used to test and confirm selected clone's specificity.Having set up " three step stainings ", used the unmarked scFv of crude extract or purification, is mouse anti myc antibody then, is at last and FITC or the bonded anti-mouse antibodies of PE.The anti-VL of the also available rabbit of this method is the anti-rabbit antibody of FITC labelling as second kind of reagent subsequently.The facs analysis of blood or bone marrow sample cell needs 5-8 * 10 ⁵The leukocyte of the individual PBS of being suspended in (containing 1%BSA).Be combined in and carried out under 4 ℃ 1 hour.After each step, washed cell also is suspended among the PBS that contains 1%BSA.Behind the final staining procedure, the final step of mensuration is erythrocytic cracking, subsequently with cell suspension in PBS, with FACS reading (Becton-Dickinson)." two step dyeing " method cell dyeing analysis can be used as the alternative of three one step process to be carried out, at first with cellular exposure in myc or biotin labeled first antibody, use anti-myc antibody of FITC labelling or PE labelling strepto-affinity uniformly dyeing color subsequently respectively.Foundation is used for facs analysis subsequently with the method for the direct cell dyeing of scFv-FITC.This new method only needs the incubation step of 1 step scFv-FITC labelling.In addition, when scFv-FITC was used for facs analysis, anti-myc significantly reduced with the high intrinsic background that normal PBL reactivity causes.Directly the result of FITC-scFv labelling and " dyeing of three steps " acquisition is quite similar, shows that the biological activity of labelling scFv is not destroyed by the FITC labeling process.Therefore, labelling scFv kept with unmarked antibody similar combine active.

Carry out the FACS scheme, analyze blood and bone marrow sample.Sample is provided by hospital, permits through the patient.The initial cell sample wherein in conjunction with scFv detection of antibodies mouse anti myc traget antibody, is finished with the anti-mice Ig of fluorescent labeling antibody then with the staining dyeing of 3 steps.In this analyzes, the results are shown in accompanying drawing 11, (N-PBL) compares with the normal circumference blood lymphocyte, tried consistent the showing with T lymphoma/leukaemia's high level of scFv (deriving from TM1.1, the TM3.13 and L32 of the elutriation of T cell) and combines.On the contrary, the B-CLL cell shows low-level scFv dyeing, to N-PBL performance similar (table 8).When the scFv dilution when 1/50 is used for facs analysis, only detect the background combination.

Table 8

Cell type	Contrast	???L32
Cell type	Contrast	???L32	T lymphoma/leukemia B-CLL N-PBL ^**	????1.4 ????0.2 ????4.9	????40 ????5 ????5

^*Anti-myc and anti-mice FITC have only been used

^*The normal circumference blood lymphocyte

In follow-up study, patient's whole blood or bone marrow sample (and sample of normal individual) are adjusted to 30 μ l/ pipe.Every pipe adds 5 μ l CD33-APC (AML) or CD19-APC (B-CLL) or CD38-APC (multiple myeloma), and every pipe also adds 5 μ l CD45-PerCp and 5 μ l scFvY1 or contrast scFv TM1.1 or CD162-PE (KPL1).Test tube is incubated 30 minutes down for 4 ℃, soft jolting.Add 2ml PBS, centrifugal 5 minutes of 1200rpm, flush away excess reagent.Supernatant discarded.In a pacing is fixed, carry out cleavage step then.500 μ l BD lysine solution ddH ₂O adds 300 μ l by dilution in 1: 10 in each patient's sample.High speed vortex oscillation sample, 4 ℃ are incubated 12 minutes, as above-mentioned washing.After the supernatant discarded, add 500 μ l PBS.Sample FACS reading is used according to international standard and is set blood specimen collection.For the mensuration that relates to 2 steps or 3 steps, working buffer liquid comprises the PBS+1%BSA+0.05% Hydrazoic acid,sodium salt, insulation and washing as above-mentioned carrying out.

For the blood sample of normal individual, analyze with FACS, to determine the comparing bonded selectivity of L32 and blood cell sub-group with the Y1 selectivity.Behind the second or the 3rd antibody staining with labelling, measure combination.All there is variability between the donor in Y1 and the L32 combination.Accordingly, represent most applications in conjunction with the qualitative table 9 that is summarized in.L32 scFv and granulocyte, lymphocyte and monocytic the combination generally are better than combining of Y1 scFv and these cells.On the contrary, L32 scFv with hematoblastic combine with background classes seemingly, generally be weaker than Y1 scFv.These results further support embodiment 6 results (referring to accompanying drawing 2), have wherein described L32 scFv to platelet aggregation among the PRP and the less influence of washing platelet coagulation.

Table 9

Cell type	The combination of negative control relatively
	The combination of negative control relatively		????Y1	????L32
	Lymphocyte mononuclear cell granulocyte platelet	Background-low+/-low-in++/-low	????Y1	????L32	Low+/-in+/ ++ in-Gao ++ background-

According to following standard grading (this is not 2 times of combinations that can be interpreted as high several times of absolute grade scale-ratio):

The dyeing of-background;

Negative scFv of+/-and the average discharge that is tried between the scFv compare≤2;

+ negative scFv and the average discharge ratio that is tried between the scFv are 2-3;

++ negative scFv and the average discharge ratio that is tried between the scFv are 4-6;

+++negative scFv and the average discharge ratio that is tried between the scFv are 6-8;

++ ++ negative scFv and the average discharge that is tried between the scFv compare greater than 10.

Should be noted that results all in the table from relating to 2 step or 3 one-step dyeing methods that signal amplifies, this point is extremely important.In addition, when the staining procedure that uses more than 1, platelet is activated, and is exaggerated from the signal of method relevant effect.In these methods, traget antibody combines with the new PSGL-1 that exposes on GPIb and the activated blood platelet.

For cancer patient's blood/bone marrow sample, compare the bonded selectivity of L32 with the Y1 selectivity with definite with facs analysis.Measure combination, be the bonded result of labelling second antibody.Described result is as shown in table 10, shows that general L32 combines with the disease cell above Y1.Simultaneously, L32 surpasses Y1 with granulocyte, lymphocyte and monocytic the combination.Reducing in conjunction with the interaction between expression and normal cell of this increase, this may be interpreted as low antibody dosage and is used for the treatment of.

Table 10

Disease	Cell type	The combination of negative control relatively
		The combination of negative control relatively		????Y1	????L32
		AML (4 samples) MM (2 samples) B leukemia (2 samples)	Disease cell lymphocyte granulocyte disease cell lymphocyte monocyte granulocyte disease cell lymphocyte monocyte granulocyte	????Y1	????L32	????+～+++ ????+～++ ????+/- ????+++ ????++ ????++ ????++ ????-～+ ????+～++ ????+++ ????++	????++++ ????+++ ????++ ????++++ ????+++ ????+++ ????+++ ????+ ????+++ ????++++ ????++++

It should be noted in all given the test agent, the combination of the anti-people PSGL1 displacement of commercial CD162 monoclonal antibody specific L32 comprises AML, hairy cell leukemia and B cell malignancies (as Pre-B-ALL, B-ALL, B-CLL, B-CLL and multiple myeloma).

Complete IgG, disome, trisome and Fab fragment all have the specificity of scFv antibody, the combination of every kind of antibody formation of anti-CD162 displacement.

Embodiment 11

Present embodiment confirms that L32 combines with each species animal primary cell.

For each species animal blood sample, estimate L32 and the bonded selectivity of blood cell sub-group.Whole blood sample is the anti-scFv of PE labelling rabbit (dyeing of 2 steps) with L32 or Y1 scFv dyeing then.Sample is analyzed with FACS subsequently.The result is as shown in table 11, shows that there is variation in being combined in of 2 kinds of antibody between species.Yet dyeing surpasses L32 to general Y1 to granulocyte, generally finds Y1 dyeing blood platelet, and L32 this two kinds of cells that do not dye show that in fact Y1 has wider specificity than L32.

Table 11

Species/cell	Combination according to facs analysis
	Combination according to facs analysis		????Y1	????L32
	Other cell of mice (Balb/c) granulocyte platelet	? ????+/- ????++ ????-	????Y1	????L32	? ????- ????- ????-
The rat all cells	Other cell of mice (Balb/c) granulocyte platelet	? ????+/- ????++ ????-	? ????-	? ????-	? ????- ????- ????-
The rat all cells	Other cell of rabbit granulocyte	? ????+ ????-	? ????-	? ????-	? ????+ ????-
Cavia porcellus lymphocyte mononuclear cell granulocyte platelet	Other cell of rabbit granulocyte	? ????+ ????-	? ????+ ????++ ????++ ????+-	? ????- ????- ????- ????-	? ????+ ????-
	The Canis familiaris L. lymphocyte	? ????-	? ????+ ????++ ????++ ????+-	? ????- ????- ????- ????-	? ????-

Mononuclear cell granulocyte platelet

???++ ???+ ???+++

????+/- ????- ????-

Embodiment 12

Present embodiment shows the effect that gives L32 to the mice that has people source malignant cell.

SCID mice (Jackson) inoculates Molt-4 (T leukemia) cell by tail cava vein injection (i.v.) with 100mg/kg CTX pretreatment, CTX injection after 5 days.Mice is divided into treatment group (6 every group) at random, and begin treatment after 5 days adopts PBS, Molt-4, Y1 treatment 3 weeks, and 2 weeks were adopted the L32 treatment.Do not treat the mice of Molt-4 group.At the 33rd day, kill mice, its liver of weighing.Accompanying drawing 12 shows that its liver weight of mice of Molt-4 growth doubles, and the tumor incidence rate of Histological determining is about 65%.Y1 or L32 scFv Antybody therapy obviously dwindle the tumor of treatment mice.

Embodiment 13

Present embodiment shows structure, expression and the purification of L32 disome and trisome.

Encode the carrier pHEN-L32 of original L32 with PCR its V that increases respectively _LAnd V _HThe district.There are MODN and antisense oligonucleotide to be used for V _LPCR reaction.The cDNA of the required size of purification, digestion with restriction enzyme is used in order-checking.With identical method amplification V _HThe district.V _HPCR product digestion with restriction enzyme.Three connection methods are used for predigestion pHEN carrier.Final carrier called after pTria-L32.After escherichia coli transform, select some clone and be used for further analysis, described analysis comprises dna sequencing, protein expression and extracts from the antibacterial periplasmic space.Under reducing condition, carry out SDS-PAGE and carry out western blot analysis, confirm the size of L32 trisome.

Handle the pTria-L32 carrier with restricted enzyme and make its linearisation, preannealing synthesizes complementary double chain oligonucleotide, is connected in the restriction enzyme site of L32 heavy chain and L32 light chain.This new support called after pDia-L32.As described in to trisome, confirm its DNA sequence and protein expression.

Expression in the escherichia coli is substantially as described in to scFv L32.But purification L32 disome and trisome are different from transformed into escherichia coli cell pericentral siphon.ScFv L32 monomeric form can be on the affinity post of protein A sepharose 4B purification.But the poly form of L32 is purification in this way, and therefore the periplasm protein that extracts from antibacterial spends the night with 60% ammonium sulfate precipitation, suspends in water, and is splined on Sephacryl-200 (Pharmacia) size-exclusion column of 0.1 * PBS pre-equilibration.Fraction collection is also analyzed with HPLC, collects the part that contains disome or trisome separately, is used for FITC labelling and facs analysis.

Embodiment 14

The production of present embodiment explanation L32-cys-kak (cysteine dimer).

1 liter of λ pL-L32-cys-kak bacterial cultures was induced 2-3 hour for 42 ℃.Centrifugal this culture is 30 minutes under the 5000rpm, and precipitation is suspended among the 180ml TE (50mM Tris-HCl pH7.4,20mM EDTA), adds in the 8ml lysozyme (5mg/ml mother solution), is incubated 1 hour.Add 20ml 5M NaCl and 25ml 25% Triton, be incubated 1 hour again.4 ℃ of centrifugal these mixture of following 13000rpm 60 minutes, supernatant discarded.The help low suspension that is deposited in historrhexis's device (or homogenizer) is in TE.This process repeats 3-4 time, is Lycoperdon polymorphum Vitt/light brown up to inclusion body (precipitation) color.

Inclusion body is dissolved in (1.5 gram inclusion bodies are dissolved in 10ml dissolving buffer, obtain about 10mg/ml soluble protein) among 6M guanidine-HCl, 0.1M Tris (pH7.4), the 2mM EDTA.This solution incubation at least 4 hours.Measure protein concentration, be adjusted to 10mg/ml.The ultimate density that adds DTE to 65mM, incubated overnight under the room temperature.10ml albumen is diluted in the solution that (dropping) contains 0.5M arginine, 0.1M Tris pH8,2mM EDTA and 0.9mM GSSG, the beginning refolding.Refolding solution is incubated 48 hours down at about 10 ℃.Contain proteic refolding solution and containing 25mM phosphate and 100mM carbamide, pH dialyses in 6 the buffer, is concentrated into 500ml.Solution through concentrating/dialysing combines with the SP agarose column, albumen gradient NaClo (the highest 1M) eluting.

Embodiment 15

Total length L32 IgG antibody and Fab ₁And F (ab ') ₂Segmental production such as following carrying out, the production of Y1IgG is herewith carried out.

CHO ^-Cell 5%CO ₂In be incubated at the F-12 culture medium that has added 10% hyclone and 40 μ g/ml gentamycins under 37 ℃.Before the transfection 1 day, 1-1.5-1 * 10 ⁶Individual cell inoculation is in the 90mm culture dish.Culture is with the DNA cotransfection of 10 μ g coding L32 light chain of antibody and heavy chain.Transfection is carried out with FuGene (Roche) transfection reagent technology.Growth is after 2 days in the non-selective growth medium, and cell was cultivated 10-12 days in the F-12 culture medium that contains 550 μ g/ml neomycin and 3 μ g/ml puromycins.Use trypsin digestion and cell, by limiting dilution to 0.5 cells/well clone cell on Costar 96 hole plastic plates.Select single clone, on 6 orifice plates, grow, transfer in the culture bottle with further selection (determining expression and the level that is secreted into antibody in the growth medium).

CHO ^-Cell 5%CO ₂In be incubated at the F-12 culture medium that has added 10% hyclone and 40 μ g/ml gentamycins under 37 ℃.Before the transfection 1 day, 0.8-1 * 10 ⁶Individual cell inoculation is in the 90mm culture dish.Culture is cloned in L32 light chain of antibody and the heavy chain and the dhfr gene that is cloned under the sv-40 promoter under CMV (cytomegalovirus) promoter with 10 μ g DNA transfections, described dna encoding.Transfection is carried out with FuGene (Roche) transfection reagent technology.Growth is after 2 days in the non-selective growth medium, and cell is cultivated in the culture medium that contains 100nM-5 μ M methotrexate (MTX), and the clone (limiting dilution after) of hyclone to select total length L32 antibody expression level to raise removed in dialysis.

Set up the sandwich ELISA algoscopy and be secreted into the concentration of the antibody in the supernatant to determine transfection CHO cell.Be quantitative antibody concentration, use following reagent: monoclonal anti human IgG1 (Fc) is (Sigma) as coated antibody, and the anti-human IgG of goat (γ chain specificity) biotin junctional complex is as detection molecules (Sigma), and the human IgG1 λ (Sigma) of purification is as standard.

Cell rolls bottle and grows to final concentration 1-2 * 10 in the F-12 culture medium that adds 10% hyclone, neomycin and puromycin (as above-mentioned) ⁸Individual cell/bottle.For antibody producing, cultured cell in same medium, but cultivated again 2 days with 2% hyclone.Excretory antibody is gone up purification at Protein G agarose column (Pharmacia) and ion exchange column-Q agarose (Pharmacia).Be combined in the 20mM sodium phosphate buffer of pH7.0 and carry out, be eluted in the 0.1M glycine buffer of pH2.5-3.0 and carry out.The amount of antibody purification is measured with UV absorbance and ELISA, and purity is analyzed with SDS-PAGE and HPLC.

L32 IgG is cracked into Fab ₁And F (ab ') ₂At first, the antibody fragment for preparing unit price and bivalence with immobilization ficin (Pierce).The splitting action of ficin in the presence of the 1mM cysteine produces F (ab ') ₂Fragment.Alike ground increases to 10mM by the cysteine activator that will digest in the buffer, can obtain the Fab fragment from original I gG.After the digestion, fragment is purification on immobilization protein A post.F (ab ') ₂And Fab ₁Fragment concentrates with microconcentrator, and molecular cut off is made as 10,000 or 30,000 dalton.Absorbance measurement protein recovery with the 280nm place.Fragment purity is measured with gel electrophoresis.

Further in the digestion buffer, handled 10mg purification L32 antibody 16 hours with 37 ℃ in 0.5ml immobilized papain slurry.With 1.5ml binding buffer liquid eluting digest, stopped reaction.Never digest in IgG and the Fc fragment with protein A post and binding buffer liquid and to separate Fab1.Fab1 is included in the mobile phase.By reading 280nm place absorbance, merge and comprise segmental peak part, concentrate, with the PBS dialysed overnight of pH7.4.Measure protein recovery, purity and character by 280nm place absorbance and gel electrophoresis.

Embodiment 16

Estimate scFv L32 to scFv Y1 and the bonded influence of ML2 cell (table 11) or to IgG Y1 and the bonded influence of ML2 cell (table 12) by the competitive assay that uses facs analysis.

Add 1 μ g competition antibody (L32 scFv, Y1 scFv (P03), KPL-1 or contrast N06 scFv) to the ML2 cell and (measure 0.5 * 10 at every turn ⁶Cell).Be incubated after 30 minutes, add the scFv or the IgG of Y1-PE labelling, again 37 ℃ of insulations 30 minutes.Insulation back sample with the washing of FACS buffer is once analyzed then.

The result is expressed as geometrical mean, and described meansigma methods is that 2 test tubes are analyzed doubles intermediate value average as a result.Geometrical mean is that the index of binding affinity is represented but not linear expression.

The result shows shown in the table 12, and L32 scFv suppresses (about 80%) scFv Y1-PE effectively and combines with the ML2 cell, and promptly L32 scFv plays inhibitory action in the mode similar to Y1 scFv.On the contrary, irrelevant negative control antibody scFv N06 only suppresses 20% combination.

Table 12

Sample	Intermediate value	Suppress percentage ratio
Sample	Intermediate value	Suppress percentage ratio	Contrast KPL-1-PE Y1-PE Y1-PE+N06 Y1-PE+P03 Y1-PE+L32	??5 ??700 ??240 ??190 ??48 ??40	????- ????- ????- ????20 ????80 ????83

Table 13

Sample	Intermediate value	Suppress percentage ratio
	Intermediate value	Suppress percentage ratio	??200ng????500ng???1000ng	?200ng??500ng???1000ng
	Contrast Y1 Y1-PE+Y1 Y1-PE+KPL-1 Y1-PE+P03 Y1-PE+L32	????3???????ND??????ND ????12.5????ND??????ND ????3???????ND??????ND ????2???????ND??????ND ????7.5?????6???????5 ????5.5?????3.8?????3.7	??200ng????500ng???1000ng	?200ng??500ng???1000ng	?-??????ND??????ND ?-??????ND??????ND ?100％??ND??????ND ?100％??ND??????ND ?40％???52％????60％ ?54％???79％????80％

Therefore ND=does not experimentize owing to be issued to inhibition fully at low concentration.

The result shows shown in the table 13, IgG Y1 (ultimate density 200ng) separately is 12.5 with the bonded geometrical mean of cell, and the competition of 200ng " cold " IgG Y1 or KPL-1 (the anti-PSGL1 mouse monoclonal antibody of commercialization supply) reduces the bonded geometrical mean to 3 of IgG Y1 and 2 respectively.IgG Y1 suppresses (competition) with combining by scFv Y1 antibody of ML2 cell in dose-dependent mode, and promptly the scFv Y1 antibody of 200ng reduces the bonded geometrical mean to 7.5 of IgG Y1, and 1000ng reduces its geometrical mean to 5.IgG Y1 is suppressed (competition) by scFv L32 antibody in dose-dependent mode with combining also of ML2 cell, but the inhibition degree surpasses scFv Y1, the scFv L32 antibody that is 200ng reduces the bonded geometrical mean to 5.5 of IgG Y1, and 1000ng reduces its geometrical mean to 3.7.

Though scFv Y1 and scFv L32 suppress combining of IgG Y1 and ML2 cell, under identical antibody concentration, the inhibition that the rejection ratio scFv Y1 that scFv L32 produces produces is more remarkable.In addition, tried scFv antibody and compare with 2, tried IgG antibody (IgG Y1 and KPL-1) and all have higher affinity with the ML2 cell, they reduce the bonded geometrical mean to 3 of IgG Y1 and 2 respectively.

Embodiment 17

Present embodiment confirms combining of L32 and various blood cell sub-groups with facs analysis, comprises normal cell (table 14) and disease cell (table 15).

At first, analyze, measure and compare L32 scFv and the bonded selectivity of CD34+ precursor with Y1 scFv, described cell is from normal person's bone marrow (NBM) sample.Measure combination with traget antibody (anti-scFv-PE) dyeing back.In same measured with each NBM sample of 2 kinds of antibody tests.

It is as follows to estimate bonded standard:

Geometrical mean (GM) is lower than the 10-feminine gender;

Geometrical mean (GM) between 11-20-the low-affinity combination;

Geometrical mean (GM) between 21-40-medium affinity combination;

Geometrical mean (GM) more than 41 and 41-the high affinity combination.

As shown in table 14,32 are tried in the NBM sample, and 24 (75%) does not combine with L32 or Y1.6 samples (19%) show the low-affinity combination to L32, and 1 (3%) shows medium affinity combination to L32, and 1 (3%) is to L32 and Y1 and show medium affinity combination.Generally speaking, L32 is similar to the bonded characteristic of CD34+ cell with Y1.

Table 14

Numeral	Code name	P03	L32	Numeral	Code name	P03	L32
Numeral	Code name	P03	L32	Numeral	Code name	P03	L32	????N1 ????N2 ????N3 ????N4 ????N5 ????N6 ????N7 ????N8 ????N9 ????N10 ????N11 ????N12 ????N13 ????N14 ????N15 ????N16	??105351 ??AH/NBM01 ??AH/NBM03 ??AH/NBM04 ??KBM33 ??AH/NBM08 ??AH/NBM09 ??AH/NBM10 ??AH/NBM11 ??AH/NBM12 ??AH/NBM13 ??105377 ??AH/NBM14 ??AH/NBM20 ??AH/NBM22 ??AH/NBM23	Negative negative 10 10 negative negative 9 is negative	Negative 13 negative 14 negative 13 14 negative negative 16 is negative	?N17 ?N18 ?N19 ?N20 ?N21 ?N22 ?N23 ?N24 ?N25 ?N26 ?N27 ?N28 ?N29 ?N30 ?N31 ?N32	?AHSBM24 ?AHJBM26 ?AHSBM27 ?AHSBM28 ?AHJBM31 ?AHJBM36 ?AHJBM37 ?AHJBM38 ?AHJBM39 ?AHJBM40 ?AHJBM41 ?AHJBM42 ?AHJBM43 ?AHJBM44 ?AHJBM45 ?102546	Negative 9.6 negative 26 is negative negative negative	Negative 22 17 28 is negative negative negative

The geometrical mean of the value representative sample in the table.

In addition, L32 scFv and Y1 scFv (PO3) are combined with isolating people's primary leukemia cell (AML, MM, B-CLL and B-ALL) from patient's blood sample carry out comparative facs analysis.The combination of in the same sample of same mensuration, comparing L32 and Y1.Combined standard is not fixed as above-mentioned.

Table 15 expression is tried the data that patient's sample obtains from all, and table 16 has been summed up the affinity data of L32 and Y1 in AML and the B-CLL sample.

Table 15

Numbering	Type	Cell type
		Cell type	Disease cell lymphocyte mononuclear cell granulocyte

L32 combines with the leukaemic's

Numbering	Type	Cell type
		Cell type					The disease cell	Lymphocyte	Mononuclear cell	Granulocyte
		????1 ????2 ????3 ????4 ????5 ????6 ????7 ????8 ????9 ????10 ????12 ????13 ????14 ? ????15 ????16 ? ????17 ????18 ????19 ????20 ????21 ????22 ????23 ????24 ????25 ????26 ????27 ????28 ????29 ????30	?42824 ?42841 ?42868 ?42873 ?42874 ?42902 ?42933 ?42939 ?42946 ?R0298 ?R9849 ?KAM095 ?R5440-7 ? ?R0376 ?KAM096 ? ?KAM108 ?KAM109 ?KMM097 ?42934 ?42938 ?KPC105 ?KBC098 ?KBC100 ?KBC101 ?KBC102 ?KBC103 ?KBC104 ?KBC105 ?KBC106	AML/M2 AML/M4 AML/M2 AML/M4 AML/M4 AML/M6 AML/M5 AML/M2 AML/M0 AML/M0 AML/M4 RAEB AML/M3 AML/M1/M 2 AML/M2 recurrence AML AML/M5 MM MM MM plasmacytoma B-CLL B-CLL B-CLL B-CLL B-CLL B-CLL B-CLL B-CLL	????53/1000 ¹330 43 24 negative 26 200 57 negative 23 470 30 200 102 36 94³91 100 650 550 340 37 10 feminine genders⁴Negative 13 feminine genders ⁴70 feminine genders	??68 ??123 ??120 ??94 ??73 ??78 ??18 ??110 ??98 ??260 ??260 ??83 ??140 ? ??65 ??41 ? ??143 ??18 ??27 ??130 ??110 ??330 ??154 ??46 ??140 ??- ??46 ??60 ??250 ??68	The disease cell	Lymphocyte	Mononuclear cell	Granulocyte	??- ??900 ²??1000 ??- ??35 ??- ? ??- ??- ??- ??- ??- ??- ? ??295 ??- ? ??360 ??- ??- ??560 ??360 ??- ??- ??- ??280 ??- ??300 ??260 ??- ??200	-280 345 80 feminine genders-160--340 240-186-280 38-150 160 280 240 68 190 110 230 negative 260 140

Numbering type	Cell type
	Cell type				The disease cell	Lymphocyte	Mononuclear cell	Granulocyte
	????31 ????32 ????33 Y1?scFv ????1 ????2 ????3 ????4 ????5 ????6 ????7 ????8 ????9 ????10 ????12 ????13 ????14 ? ????15 ? ????16 ????17 ? ????18 ????19 ????20 ????21 ????22 ? ????23 ????24 ????25 ????26 ????27	KBC107 B-CLL R3093-7 B-ALL R313-0 B-ALL and leukaemic's recurs AML KAM109 AML/M5 KMM097 MM 42934 MM 42938 MM KPC105 plasmacytoma KBC098 B-CLL KBC100 B-CLL KBC101 B-CLL KBC102 B-CLL KBC103 B-CLL in conjunction with 42824 AML/M2,42841 AML/M4,42868 AML/M2,42873 AML/M4,42874 AML/M4,42902 AML/M6,42933 AML/M5,42939 AML/M2,42946 AML/M0 R0298 AML/M0 R9849 AML/M4 KAM095 RAEB R5440-7 AML/M3 R0376 AML/M1/M, 2 KAM096 AML/M2 KAM108	17 negative negative/87 ¹125 15 12 negative 19 62 8 negative 230 12 95 29 17 15³40 70 170 184 117 21 19 feminine genders⁴Negative	??165 ??41 ??26 ? ??50 ??59 ??53 ??38 ??36 ??24 ??10 ??18 ??27 ??43 ??160 ??20 ??43 ? ??18 ? ??15 ??20 ? ??14 ??13 ??30 ??30 ? ??116 ??70 ??17 ??29 ??- ??20	The disease cell	Lymphocyte	Mononuclear cell	Granulocyte	??420 ??490 ??- ? ? ??270 ²??100 ??- ??30 ??- ? ??- ??- ? ??- ??- ? ? ??100 ? ??- ??110 ? ??- ??- ??160 ??128 ? ??- ??- ??- ??84 ??- ??86	270 90 100 126 180 43 feminine genders-51-225 74 63-120 18-50 68 100 140 27 47 60 110

Numbering	Type	Cell type
		Cell type				The disease cell	Lymphocyte	Mononuclear cell	Granulocyte
		????28 ????29 ????30 ????31 ????32 ????33	?KBC104 ?KBC105 ?KBC106 ?KBC107 ?R3093-7 ?R313-0	?B-CLL ?B-CLL ?B-CLL ?B-CLL ?B-ALL ?B-ALL	Negative ⁴46 negative 9.5 is negative	The disease cell	Lymphocyte	Mononuclear cell	Granulocyte	????10 ????117 ????41 ????79	????84 ????- ????98	Negative 133 65 126

1 two kinds of diseases;

In the 2 disease cells;

3 contain the peripheral blood of 2.5%CD34+ cell;

4 cells are CD19+/CD5-

17 kinds are tried in the AML sample, and 15 kinds (88%) combine the positive with L32, and wherein 10 kinds of samples (59%) show high affinity in conjunction with (average geometric meansigma methods 165), and 5 kinds of samples (29%) show medium affinity in conjunction with (average geometric meansigma methods 28).It is also positive that 13 kinds of (76%) AML samples combine with Y1, but comparative analysis (table 16) shows, the average binding affinity of L32 is always apparently higher than the average binding affinity of Y1 in the same sample.In general, about 50% M2 phase AML patient sample shows and can combine with L32 and Y1.In M3 and more late period, about 90% sample can with 2 kinds of antibodies, its affinity degree have nothing in common with each other (data not shown).

9 kinds are tried in the B-CLL sample, 4 kinds (44%) combine the positive with L32, wherein a kind of sample (11%) shows high affinity in conjunction with (geometrical mean 70), 1 kind of sample (11%) shows medium affinity in conjunction with (geometrical mean 37), and 2 kinds of samples (22%) show low-affinity in conjunction with (average geometric meansigma methods 15).3 kinds (33%) is tried the B-CLL sample and is combined also positively with Y1, but comparative analysis (table 16) shows, the average binding affinity of L32 is usually apparently higher than the average binding affinity of Y1 in the same sample.

4 kinds are tried in MM and the plasmocytoma sample, and all samples shows with the high affinity of L32 and Y1 and combines, but the affinity of L32 is usually apparently higher than the affinity (the average geometric meansigma methods is respectively 410 and 135) of Y1.2 kinds are tried in the B-ALL sample, and 2 kinds all negative with combining of L32 and Y1.

Table 16

???????AML	????B-CLL
???????AML	????B-CLL	????L32????Y1	??L32???Y1
The medium geometrical mean disease of high geometrical mean disease cell lymphocyte monocyte granulocyte cell lymphocyte monocyte granulocyte hangs down geometrical mean disease cell lymphocyte monocyte granulocyte	165 61 117 50 638 145 239 110 28 12 41 15 35 30 negative nothing 18 12 feminine genders are negative negative	????L32????Y1	??L32???Y1	70 46 107 42 323 58 169 70 37 21 26 9 negative 13 9 is negative negative

^*The average geometric meansigma methods is based on table 14 result.High, medium and low-affinity group is determined according to the L32 data.

Result in the table 15 and 16 clearlys show, in the disease cell and other mature cell subgroup (lymphocyte, mononuclear cell and granulocyte) of former generation AML and B-CLL blood sample, the combination of L32 antibody is usually apparently higher than the combination of Y1.

Sequence table

<110>Bio-Technology?General?Corp

＜120〉L32 antibody and application thereof

<130>10793/63

<140>10/189,032

<141>07/01/2002

<160>8

<170>FastSEQ?for?Windows?Version?3.0

<210>1

<211>280

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>1

Met?Lys?Tyr?Leu?Leu?Pro?Thr?Ala?Ala?Ala?Gly?Leu?Leu?Leu?Leu?Ala?Ala?Gln?Pro?Ala

1???????????????5???????????????????10??????????????????15??????????????????20

Met?Ala?Glu?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Arg?Pro?Gly?Gly?Ser?Leu

25??????????????????30??????????????????35??????????????????40

Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Asp?Leu?Asn?Pro?Lys?Val?Lys?His?Met

45??????????????????50??????????????????55??????????????????60

Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val?Ser?Gly?Ile?Asn?Trp?Asn?Gly

65??????????????????70??????????????????75??????????????????80

Gly?Ser?Thr?Gly?Tyr?Ala?Asp?Ser?Val?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ala

85??????????????????90??????????????????95??????????????????100

Lys?Asn?Ser?Leu?Tyr?Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr

105?????????????????110?????????????????115?????????????????120

Cys?Ala?Arg?Met?Arg?Ala?Pro?Val?Ile?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Arg

125?????????????????130?????????????????135?????????????????140

Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Ser?Glu?Leu?Thr?Gln

145?????????????????150?????????????????155?????????????????160

Asp?Pro?Ala?Val?Ser?val?Ala?Leu?Gly?Gln?Thr?Val?Arg?Ile?Thr?Cys?Gln?Gly?Asp?Ser

165?????????????????170?????????????????175?????????????????180

Leu?Arg?Ser?Tyr?Tyr?Ala?Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Val?Leu?Val

185?????????????????190?????????????????195?????????????????200

Ile?Tyr?Gly?Lys?Asn?Asn?Arg?Pro?Ser?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Gly?Ser?Ser?Ser

205?????????????????210?????????????????215?????????????????220

Gly?Asn?Thr?Ala?Ser?Leu?Thr?Ile?Thr?Gly?Ala?Gln?Ala?Glu?Asp?Glu?Ala?Asp?Tyr?Tyr

225?????????????????230?????????????????235?????????????????240

Cys?Asn?Ser?Arg?Asp?Ser?Ser?Gly?Asn?His?Val?Val?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Thr

245?????????????????250?????????????????255?????????????????260

Val?Leu?Gly?Ala?Ala?Ala?Glu?Gln?Lys?Leu?Ile?Ser?Glu?Glu?Asp?Leu?Asn?Gly?Ala?Ala

265?????????????????270?????????????????275?????????????????280

<210>2

<211>6

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>2

Met?Arg?Ala?Pro?Val?Ile

5

<210>3

<211>16

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>3

Gly?Ile?Asn?Trp?Asn?Gly?Gly?Ser?Thr?Gly?Tyr?Ala?Asp?Ser?Val?Lys

1???????????????5???????????????????10??????????????????15

<210>4

<211>8

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>4

Leu?Asn?Pro?Lys?Val?Lys?His?Met

1???????????????5

<210>5

<211>7

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>5

Leu?Arg?Gly?Gly?Asn?Ala?Met

1???????????????5

<210>6

<211>11

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>6

Phe?Leu?Thr?Tyr?Asn?Ser?Try?Glu?Val?Pro?Thr

1???????????????5???????????????????10

<210>7

<211>9

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>7

Thr?Asn?Trp?Tyr?Leu?Arg?Pro?Leu?Asn

1???????????????5

<210>8

<211>10

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>8

Glu?Gln?Lys?Leu?Ile?Ser?Glu?Glu?Asp?Leu

1???????????????5???????????????????10

Claims

1. antibody or its fragment in conjunction with a PSGL-1 epi-position, wherein said antibody or its fragment have the binding ability of SEQ ID NO:1 scFv.

2. the antibody of claim 1 or its fragment, wherein said antibody or its fragment comprise the heavy chain complementary determining region (CDR) of a kind of SEQ of being selected from ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

3. the antibody of claim 2 or its fragment, wherein 2 kinds of heavy chain CDR are selected from SEQ IDNO:2, SEQ ID NO:3 and SEQ ID NO:4.

4. the antibody of claim 3 or its fragment, wherein 3 kinds of heavy chain CDR are selected from SEQ IDNO:2, SEQ ID NO:3 and SEQ ID NO:4.

5. claim 4 antibody or its fragment, wherein said antibody or its fragment comprise SEQ IDNO:1.

6. antibody or its fragment in conjunction with a PSGL-1 epi-position, wherein said antibody or its fragment comprise the heavy chain complementary determining region (CDR) of a kind of SEQ of being selected from ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

7. the antibody of claim 6 or its fragment, wherein 2 kinds of heavy chain CDR are selected from SEQ IDNO:2, SEQ ID NO:3 and SEQ ID NO:4.

8. the antibody of claim 7 or its fragment, wherein 3 kinds of heavy chain CDR are selected from SEQ IDNO:2, SEQ ID NO:3 and SEQ ID NO:4.

9. antibody or its fragment in conjunction with a PSGL-1 epi-position, described antibody or its fragment comprise SEQ ID NO:1.

10. the antibody of claim 1 or its fragment, wherein said antibody or its fragment comprise the skeleton variable region that at least one derives from DP32 kind system.

11. the antibody of claim 1 or its fragment, wherein said antibody or its fragment are circular or annular peptide or polypeptide.

12. claim 1 antibody or its fragment, wherein said epi-position comprise at least 1 sulphation part.

13. the antibody of claim 1 or its fragment, wherein said antibody or its fragment are in conjunction with 2 or multi-epitope more, and each epi-position comprises one or more sulphation tyrosine residues.

14. the antibody of claim 13 or its fragment, wherein each epi-position comprises at least 1 acidic amino acid of being made up of 2 or more a plurality of acidic amino acid bunch.

15. the antibody of claim 1 or its fragment, wherein said antibody or its fragment and 2 or more multi-epitope cross reaction, each epi-position has one or more sulphation tyrosine residues.

16. the antibody of claim 15 or its fragment, wherein each epi-position comprises at least 1 by 2 or the acidic amino acid formed of polyacid acidic amino acid bunch more.

17. the antibody of claim 1 or its fragment, wherein said antibody or its fragment combine with epi-position at least a following cell type: T-ALL cell, AML cell, B leukaemia, B-CLL and multiple myeloma cells.

18. the antibody of claim 1 or its fragment, wherein said antibody or its fragment combine with epi-position on fat, saccharide, peptide, glycolipid, glycoprotein, lipoprotein and/or the lipopolysaccharide molecule.

19. the antibody of claim 1 or its fragment, wherein said antibody or its fragment and following drug coupling or compound: anticarcinogen, antileukemia, antimetastatic agents, antineoplastic agent, disease-resistant medicine, anti-adhesive medicine, antithrombotic drug, anti-restenosis, anti-autoimmune medicine, anti-medicine, antimicrobial drug, antiviral agents and the anti-inflammatory agent assembled.

20. the antibody of claim 19 or its fragment, its Chinese medicine are the antiviral agents that is selected from acyclovir, ganciclovir and zidovudine.

21. the antibody of claim 19 or its fragment, its Chinese medicine are the antithrombotic formation/anti-restenosis that is selected from cilostazol, dalteparin sodium, Reviparin Sodium and aspirin.

22. the antibody of claim 19 or its fragment, its Chinese medicine are to be selected from the anti-inflammatory agent that former times, indomethacin, rofecoxib and nimesulide are examined in zaltoprofen, pranoprofen, drogelor, aspirin 17, diclofenac, ibuprofen, dexibuprofen, sulindac, naproxen, ammonia tolmetin, match.

23. the antibody of claim 19 or its fragment, its Chinese medicine are the anti-autoimmune medicine that is selected from leflunomide, denileukin diftitox, subreum, WinRho SDF, defibrotide and cyclophosphamide.

24. the antibody of claim 19 or its fragment, its Chinese medicine is for being selected from limaprost, cloricromen and hyaluronic anti-adhesive/anti-medicine of assembling.

25. the antibody of claim 19 or its fragment, its Chinese medicine is selected from toxin, radiosiotope, preparation and medical substance.

26. the antibody of claim 25 or its fragment, wherein toxin is selected from gelonin, Pseudomonas exotoxin (PE), PE40, PE38, ricin and their trim or derivant.

27. the antibody of claim 25 or its fragment, wherein radiosiotope is selected from γ emitting substance, positron emission thing, x ray emission thing, β emitting substance and α emitting substance.

28. the antibody of claim 25 or its fragment, wherein radiosiotope is selected from ¹¹¹Indium, ¹¹³Indium, ^99mRhenium, ¹⁰⁵Rhenium, ¹⁰¹Rhenium, ^99mTechnetium, ^121mTellurium, ^122mTellurium, ^125mTellurium, ¹⁶⁵Thulium, ¹⁶⁷Thulium, ¹⁶⁸Thulium, ¹²³Iodine, ¹²⁶Iodine, ¹³¹Iodine, ¹³³Iodine, ^81mKrypton, ³³Xenon, ⁹⁰Yttrium, ²¹³Bismuth, ⁷⁷Bromine, ¹⁸Fluorine, ⁹⁵Ruthenium, ⁹⁷Ruthenium, ¹⁰³Ruthenium, ¹⁰⁵Ruthenium, ¹⁰⁷Hydrargyrum, ²⁰³Hydrargyrum, ⁶⁷Gallium and ⁶⁸Gallium.

29. the antibody of claim 25 or its fragment, wherein medical substance is an anthracycline.

30. the antibody of claim 29 or its fragment, wherein anthracycline is selected from doxorubicin, daunorubicin, idarubicin, detorubicin, carminomycin, epirubicin, esorubicin, morpholino doxorubicin, morpholino daunorubicin and methoxy morpholine doxorubicin.

31. the antibody of claim 25 or its fragment, wherein medical substance is selected from cisplatin, taxol, calicheamicin, vincristine, cytosine arabinoside (Ara-C), cyclophosphamide, prednisone, fludarabine, chlorambucil, interferon-ALPHA, hydroxyurea, temozolomide, Thalidomide and bleomycin and their derivant and compositions.

32. the antibody of claim 19 or its fragment, wherein said antibody or its fragment and media or carrier coupling or compound, described media or carrier can with surpass a kind of drug coupling or compound.

33. the antibody of claim 32 or its fragment, wherein media or carrier are selected from glucosan, lipophilic polymer, HPMA and liposome and their derivant and trim.

34. an isolating epi-position, wherein said epi-position comprise antibody or the bonded aminoacid sequence of its fragment with claim 1.

35. the separation epi-position of claim 34, wherein said separation epi-position comprises at least one sulphation part.

36. the separation epi-position of claim 35, wherein sulphation partly is a sulphation tyrosine.

37. the separation epi-position of claim 34, wherein said separation epi-position comprise negative charge aminoacid bunch.

38. the separation epi-position of claim 37, wherein said aminoacid bunch comprise the 1-17 aminoacid of ripe PSGL-1.

39. the polynucleotide of an isolated or purified, described polynucleotide encoding claim 1 antibody or its fragment.

40. expression vector that comprises claim 39 polynucleotide sequence.

41. recombinant host cell that comprises claim 40 expression vector.

42. the recombinant host cell of claim 41 or its daughter cell, the wherein described antibody of cellular expression or its fragment.

43. a method of producing reconstitution cell, described method comprise with claim 40 expression vector transfectional cell.

44. produce antibody or its segmental method for one kind, described method is included in the cell of cultivating claim 41 under the condition that allows described antibody or its fragment expression.

45. the method for claim 44, wherein said method further comprise isolated or purified antibody or its fragment from cell or cell culture medium.

46. Pharmaceutical composition that comprises claim 1 antibody or its fragment and pharmaceutical carrier.

47. one kind be used to diagnose, prognosis prediction or test kit by stages, described test kit comprises antibody or its fragment and the preparation of claim 1.

48. the diagnosis of claim 47, prognosis prediction or test kit by stages, wherein preparation is a radiosiotope.

49. a method for the treatment of disease, described method comprises the Pharmaceutical composition that the patient of needs claim 46 is arranged.

50. a method for the treatment of cell rolling, described method comprises the Pharmaceutical composition that the patient of needs claim 46 is arranged.

51. the method for an amelioration of inflammation, prevention of inflammation, treatment inflammation or inflammation-inhibiting development, described method comprises the Pharmaceutical composition that the patient of needs claim 46 is arranged.

52. a method for the treatment of infection, described method comprises the Pharmaceutical composition that the patient of needs claim 46 is arranged.

53. the method for claim 52 wherein infects by HIV and causes.

54. the method for claim 52, wherein said administration stop HIV to enter cell.

55. a method for the treatment of autoimmune disease, described method comprises the Pharmaceutical composition that the patient of needs claim 46 is arranged.

56. a method for the treatment of transfer, described method comprises the Pharmaceutical composition that the patient of needs claim 46 is arranged.

57. a method for the treatment of growth of tumour cell and/or propagation, described method comprises the Pharmaceutical composition that the patient of needs claim 46 is arranged.

58. a method that increases the death of neoplastic cells rate, described method comprises the Pharmaceutical composition that the patient of needs claim 46 is arranged.

59. a method for the treatment of leukaemia's growth and/or propagation, described method comprises the Pharmaceutical composition that the patient of needs claim 46 is arranged.

60. a method that increases leukaemia's mortality rate, described method comprises the Pharmaceutical composition that the patient of needs claim 46 is arranged.

61. a method that changes the sensitivity of disease cell enantiopathy property of medicine damage, described method comprises the Pharmaceutical composition that the patient of needs claim 46 is arranged.

62. a method that strengthens tumor cell to the sensitivity of anticancer property of medicine damage, described method comprises the Pharmaceutical composition that the patient of needs claim 46 is arranged.

63. a method that strengthens the leukaemia to the sensitivity of anticancer property of medicine damage, described method comprises the Pharmaceutical composition that the patient of needs claim 46 is arranged.

64. the method that the tumor cell quantity that suppresses in the tumor patient increases, described method comprises the Pharmaceutical composition that the patient of needs claim 46 is arranged.

65. a method that reduces the tumor cell quantity in the tumor patient, described method comprises the Pharmaceutical composition that the patient of needs claim 46 is arranged.

66. the method that the leukaemia's quantity that suppresses among the leukaemic increases, described method comprises the Pharmaceutical composition that the patient of needs claim 46 is arranged.

67. a method that reduces the leukaemia among the leukaemic, described method comprises the Pharmaceutical composition that the patient of needs claim 46 is arranged.

68. a method that causes antibody-dependent cell-mediated cytotoxicity (ADCC), described method comprises the Pharmaceutical composition that the patient of needs claim 46 is arranged.

69. a method that stimulates natural killer cell (NK) or T cell, described method comprises the Pharmaceutical composition that the patient of needs claim 46 is arranged.

70. patient disease diagnosed or the method for prognosis prediction for one kind, described method comprises provides the sample that comprises patient's cell, whether antibody or its fragment of determining claim 1 then combine with patient's cell, show that thus the patient is described disease risks patient or suffers from described disease.

71. patient's inflammation diagnosed or the method for prognosis prediction for one kind, described method comprises provides the sample that comprises patient's cell, whether antibody or its fragment of determining claim 1 then combine with patient's cell, show that thus the patient is inflammation risk patient or suffers from inflammation.

72. the patient infection diagnosed or the method for prognosis prediction for one kind, described method comprises provides the sample that comprises patient's cell, whether antibody or its fragment of determining claim 1 then combine with patient's cell, show that thus the patient is the infection risk patient or suffers from infection.

73. the method for claim 72 wherein infects by HIV and causes.

74. patient's autoimmune disease diagnosed or the method for prognosis prediction for one kind, described method comprises provides the sample that comprises patient's cell, whether antibody or its fragment of determining claim 1 combine with patient's cell, show that thus the patient is autoimmune disease risk patient or suffers from autoimmune disease.

75. the transfer to the patient is diagnosed, prognosis prediction or method by stages, described method comprises provides the sample that comprises patient's cell, whether antibody or its fragment of determining claim 1 then combine with patient's cell, show that thus the patient shifts for shift risk patient or existence.

76. one kind to the patient tumors cell diagnose, prognosis prediction or method by stages, described method comprises provides the sample that comprises patient's cell, whether antibody or its fragment of determining claim 1 then combine with patient's cell, show that thus the patient is tumor cell risk patient or has tumor cell.

77. one kind to patient's leukemia diagnose, prognosis prediction or method by stages, described method comprises provides the sample that comprises patient's cell, whether antibody or its fragment of determining claim 1 then combine with patient's cell, show that thus the patient is leukemia risk patient or suffers from leukemia.

78. comprising, a method of eliminating the patient tumors cell, described method provide the sample that comprises patient's cell, then with antibody or its fragment incubation of patient's cell and claim 1.

79. the method for claim 78 is wherein eliminated the patient tumors cell and is finished external.

80. the purposes of the Pharmaceutical composition of claim 46 in the medicine of preparation treatment disease.

81. the purposes of claim 80, wherein disease is selected from cell rolling, inflammation, autoimmune disease, infection, transfer, growth of tumour cell and/or propagation and leukaemia's growth and/or propagation.

82. be used to prepare the Pharmaceutical composition of the claim 46 of the medicine for the treatment of disease.

83. the Pharmaceutical composition of claim 82, wherein disease is selected from cell rolling, inflammation, autoimmune disease, infection, transfer, growth of tumour cell and/or propagation and leukaemia's growth and/or propagation.

84. produce antibody or its segmental method, said method comprising the steps of for one kind:

Phage display library is provided;

At least 2 kinds and antibody or bonded molecule of its fragment or cell are provided, and described antibody or its fragment have scFv antibody or its segmental binding ability with SEQ ID NO:1;

The elutriation phage display library is selected the phage particle that shows oligopeptide or polypeptide, and described oligopeptide or polypeptide can combine with at least 2 kinds of described molecules or cell;

Produce antibody or its fragment, comprise antibody or its fragment or its binding fragment and comprise peptide or polypeptide in conjunction with at least 2 kinds of described molecules or cell.

85. antibody or its fragment that produces with the method for claim 84.