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CN1678348A - Compositions and methods for therapeutic treatment - Google Patents

Compositions and methods for therapeutic treatment Download PDF

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Publication number
CN1678348A
CN1678348A CNA03820441XA CN03820441A CN1678348A CN 1678348 A CN1678348 A CN 1678348A CN A03820441X A CNA03820441X A CN A03820441XA CN 03820441 A CN03820441 A CN 03820441A CN 1678348 A CN1678348 A CN 1678348A
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compositions
cell
antibody
medicine
fragment
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J·拉扎罗维茨
A·尼姆罗德
H·霍奇马－哈伊姆
A·莱瓦农
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Savient Pharmaceuticals Inc
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Savient Pharmaceuticals Inc
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    • C07K2317/622—Single chain antibody (scFv)

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Abstract

The present invention relates to compositions utilizing an agent and an antibody, or fragment thereof. In these compositions, the agents, including agents such as anti-cancer, anti-metastasis, anti-leukemia, anti-disease, anti-adhesion, anti-thrombosis, anti -restenosis, anti-autoimmune, anti-aggregation, anti-bacterial, anti-viral, and anti-inflammatory agents, can be complexed or combined with or conjugated to the antibodies, or fragments thereof. In addition, the agent and/or the antibody, or fragment thereof, can be present in the composition in a sub-clinical amount, which is an amount that is less than the amount of the agent generally found to be clinically effective when the agent is administered alone. Preferably, in these compositions of the present invention, the agent is an anthracycline or a derivative thereof, e.g., doxorubicin (adriamycin) or a derivative thereof.

Description

The compositions of being used for the treatment of property treatment and method
Invention field
The present invention relates to therapeutic combination and diagnosis composition and method, described compositions and method utilization have active anticancer, antimetastatic activity, anti-leukocythemia liveness, antiviral activity, the medicine and the antibody of anti-infection activity and/or anti-other disease activity, described other disease is inflammatory diseases for example, the disease that relates to unusual adhesion or pathogenic adhesion, thrombosis and/or restenosis, relate to unusual gathering or pathogenic accumulative disease and autoimmune disease, cardiovascular disease is myocardial infarction for example, retinopathy, by sulphation tyrosine dependence protein matter the interact disease cause and common diseased cells.
Background of invention
Antibody, phage display and tissue target to
The tissue selectivity targeting of curative is the emerging direction in the pharmaceuticals industry.Designed new cancer therapy,, reduced toxicity simultaneously and improve general curative effect to increase the specificity and the effectiveness of treatment based on targeting.Be used for trial at the mouse monoclonal antibody (MAb) of tumor associated antigen with toxin, radioactive nucleus thuja acid and chemotherapy conjugate target tumor.In addition, developed differentiation such as CD19, CD20, CD22 and CD25 antigens in treatment hemopoietic malignant tumor as the cancer specific target.Though carried out deep research, this method still has several places limitation.A limitation is the monoclonal antibody that is difficult to separate suitable displaying selective binding.Second limitation is to need the prerequisite of high antibody mediated immunity originality as successful separation antibody.The 3rd limitation is that final products include the non-human sequence, the feasible immunne response (for example human anti-mouse antibody-HAMA replys) that produces the non-human material.This HAMA replys short serum half life of frequent generation and stops repetitive therapy, thereby has reduced the therapeutic value of antibody.This succeeding switch is sex-limited to have evoked people in this interest aspect two of Mus source engineering chimeric mAb or Humanized monoclonal antibodies and finder's antibody.Another limitation of this method is that it only can separate only at known and a kind of antibody purifying antigen.And this method does not have selectivity to allowing to separate at the antibody that is present in the anti-cell surface markers on normal cell and the malignant cell.
Many factors that influence mab treatment treatment for cancer effect are arranged.These factors comprise the accessibility of antigenic specificity, expression, antigenic heterogeneity and the tumor mass of tumor cells expression.Leukemia and lymphoma have more reactivity than solid tumor such as cancer usually for the treatment of antibody.Monoclonal antibody promptly is attached to leukaemia and the lymphoma cell in the blood flow, and easily is penetrated into the malignant cell in the lymphoid tissue, thereby lymph tumor becomes the fabulous candidate based on mab treatment.An ideal system must be able to identify that identification produces the monoclonal antibody of the stem cell cell surface marker of pernicious daughter cell.
Phage library can be used for random screening and is attached to isolating, the predetermined target protein such as the strand Fv (scFv) of antibody, hormone and receptor.In addition, use the antibody display libraries usually, phage scFv library particularly can be impelled and be found to be used for targeting specific, do not differentiate the alternative method of the unique molecular of the undetermined cell surface part of Buddhist monk as yet.
Leukemia, lymphoma and myeloma are derived from bone marrow and lymphoid tissue and the cancer that relates to the uncontrolled growth of cell.Acute lymphoblastic leukemia (ALL) is the different substantiality disease according to specific Clinical symptoms and amynologic characteristic definition.ALL form with other is the same, though the definite cause of disease of most B cell ALL (B-ALL) case is unknown, in many cases, this disease makes cell become unusual and continuous propagation by due to the homotropic inheritance change of unicellular DNA.Suffer from the patient's of B-ALL prognosis, among both, all significantly poorer child and adult than suffering from other leukemic patient.
Acute myeloid leukaemia (AML) is the heterogeneous tumor colony that produces the CFU-GM of the whole myeloid lineage cell (erythrocyte, granulocyte, mononuclear cell and platelet) of end differentiation under normal operation.The same with other tumor form, AML and homotropic inheritance change relevant, and the undifferentiated blastocyte relatively that it causes the medullary cell of normal differentiation to be shown one or more early stage bone marrow differentiation types is replaced.Usually AML is in bone marrow and developing in the secondary hemopoietic organ on the littler degree.AML mainly influences the adult, and maximum effect scope is the people of 15-40 year age bracket, but same known it not only influence the child but also influence the old people.Nearly all AML patient need treat after diagnosis to reach clinical remission immediately, does not wherein have circulation not break up the evidence of blastocyte abnormal level.
Up to now, developed the monoclonal antibody of the dissolved cell activity of multiple inducing antitumor cell.FDA has ratified the humanization form MuMAb4D5 at the monoclonal antibody of P185-growth factor receptors (HER2) ectodomain, and uses it for treatment human breast carcinoma (United States Patent (USP) the 5th, 821, No. 337 and the 5th, 720, No. 954).After combination, described antibody can suppress to depend on the growth of tumour cell of HER2 growth factor receptors.In addition, resist to cause the chimeric antibody that comprises the CD20 that those are relevant with lymphoma that periphery B cell exhausts fast, in the recent period by FDA approval (United States Patent (USP) the 5th, 843, No. 439).This antibody produces the complement-dependent lysis with combining of target cell.This product is granted in the recent period and be used for the treatment of rudimentary B cell non-Hodgkin's at present clinically.
Among some other humanized antibody and chimeric antibody are being developed or carrying out clinical trial.In addition, on the normal marrow cell surface but also at the humanization Ig of the CD33 antigenic specificity reaction of the marrow leukaemia cell surface expression of most of types, can put together (Sievers etc., Blood supplementary issue with not only with anticarcinogen calicheamicin CMA-676,308,504a (1997)).This conjugate is commonly called as medicine MYLOTARGT , obtained the approval (Caron etc., Cancer supplementary issue, 73,1049-1056 (1994)) of FDA in the recent period.According to its dissolved cell activity, another is the anti-CD 33 antibody (HumM195) in clinical trial in the recent period, can put together with several cytotoxic agents, comprise and gelonin (McGraw etc., Cancer Immunol.Immunother, 39,367-374 (1994)) and radiosiotope 131I (Caron etc., Blood83,1760-1768 (1994)), 90Y (Jurcic etc., Blood supplementary issue, 92,613a (1998)) and 213Bi (Humm etc., Blood supplementary issue, 38:231P (1997)) puts together.
A kind of chimeric antibody (cHuLym3) that is used for the treatment of human leukemia and lymphadenomatous anti-human leucocyte antigen CD45 is carrying out clinical research (Sun etc., Cancer Immunol.Immunother., 48,595-602 (2000)).In experiment in vitro, in ADCC (cytotoxicity of antibody dependent cellular mediation) experiment, observe specificity lysis (Henkart, Immunity, 1,343-346 (1994); Squier and Cohen, Current Opin.Immunol., 6,447-452 (1994)).
Opposite with the structure of mouse monoclonal humanized antibody and chimeric antibody, use display technique of bacteriophage and can separate the scFv that comprises complete human sequence.Exploitation one strain is recently cloned based on scFv, comes from the fully human antibodies of the anti-people TGFb2 receptor of display technique of bacteriophage.This being switched in the complete human IgG 4 can be competed bonded scFv (Thompson etc., J.Immunol Methods, 227,17-29 (1999)) with TGFb2 and be had extremely strong antiproliferative activity.This technology well known by persons skilled in the art more specifically is described in the following publication: Smith, Science, 228,1315 (1985); Scott etc., Science, 249,386-390 (1990); Cwirla etc., PNAS, 87,6378-6382 (1990); Devlin etc., Science, 249,404-406 (1990); Griffiths etc., EMBO J., 13 (14), 3245-3260 (1994); Bass etc., Proteins, 8,309-314 (1990); McCafferty etc., Nature, 348,552-554 (1990); Nissim etc., EMBO J., 13,692-698 (1994); United States Patent (USP) the 5th, 427, No. 908, the 5th, 432, No. 018, the 5th, 223, No. 409 and the 5th, 403, No. 484, lib..
The part of isolating scFv antibody molecule
Platelet, fibrinogen, GPIb, selection albumen and P selection protein sugar protein ligands-1 (PSGL-1) wait in each comfortabler disease or for example unusual or pathogenic inflammation of morbid state, unusual or pathogenic immunoreation, autoimmune response, transfer, unusual or pathogenic adhesion, thrombosis and/or restenosis and the unusual or pathogenic gathering and play an important role.Therefore, the antibody with platelet and these molecule cross reactions will can be used for diagnosing and treating disease and the obstacle that comprises these and other pathogenic disease.
Platelet
Platelet is a blood system typical characteristic composition, and plays an important role in hemostasis, thrombosis and/or restenosis and restenosis.Damage to blood vessel is in the running for example known hemostasis, it is characterized in that a series of continuous incidents.Be subjected to the invasion and attack district to what the initial action of blood vessel of damage was a thrombocyte adhesiveness to vascular inner surface.Next step be the multilamellar platelet aggregation on the platelet of previous adhesion, form tampon.This platelet is certainly sealed described blood vessel wall.This tampon is strengthened because of the deposition of fibrin polymer.This blood clot only just is degraded when described damage is repaired.
The importance of platelet in transfer
Neoplasm metastasis perhaps is the greatest factor of restriction cancer patient existence.The data that accumulated show that tumor cell and the interactional ability of host's platelet are to shift one of indispensable determiner.Leslie Oleksowicz, Z.M., " Characterization Of Tumor-Induced Platelet Aggregation:The Role Of Immunorelated GPIb AndGPIIb/IIIa Expression By MCF-7 Breast Cancer Cells (the platelet aggregation feature of tumor promotion: relevant GPIb of immunity that expresses by the MCF-7 breast cancer cell and the effect of GPIIb/IIIa), " Thrombosis Research 79:261-274 (1995).
It is relevant to have proved that tumor cell is assembled the metastatic potential of hematoblastic ability and tumor cell, and has shown that the platelet aggregation that suppresses tumor promotion is relevant with metastasis inhibition in the rodent model.Proved that tumor cell and hematoblastic interaction relate to the secretion of film adhesion molecule and agonist.The expression of the relevant platelet glycoprotein of immunity is identified in tumor cell line.The Ia glycoprotein GPIb of platelet, GPIIb/IIIa, GPIb/IX and beta 2 integrin alpha have been proved vSubunit is at the breast tumor cell line surface expression.Oleksowicz, Z.M., " Characterization Of Tumor-Induced Platelet Aggregation:TheRole Of Immunorelated GPIb And GPIIb/IIIa Expression By MCF-7Breast Cancer Cells (the platelet aggregation feature of tumor promotion: relevant GPIb of immunity that expresses by the MCF-7 breast cancer cell and the effect of GPIIb/IIIa), " Thrombosis Research79:261-274 (1995); Kamiyama, M etc., " Inhibition of platelet GPIIb/IIIabinding to fibrinogen by serum factors:studies of circulating immunecomplexes and platelet antibodies in patients with hemophilia; immunethrombocytopenic purpura; human immunodeficiency virus-relatedimmune thrombocytopenic purpura; and and systemic lupus erythematosus (serum factor is to the inhibition in conjunction with the platelet GPIIb/IIIa of fibrinogen: hemophilia; immunologic thrombocytopenic purpura; the research of the immunologic thrombocytopenic purpura that the human immunodeficiency virus is correlated with and Patients with SLE body-internal-circulation immune complex and platelet antibody), and " J Lab Clin Med 117 (3): 209-17 (1991).
Gasic (J.T.B.Gasic etc., Proc.Natl.Acad.Sci.USA 61:46-52 (1968)) and colleague proof, the thrombocytopenia that antibody brings out reduces the number and the volume of the metastatic tumor that is produced by CT26 adenocarcinoma of colon, lewis lung cancer and B16 melanoma significantly.Karpatkin etc., " Role of adhesive proteins in platelet tumor interaction in vitro andmetastasis formation in vivo (effect of adhesion protein in external interaction of platelet tumor and the external transfer formation), " J.Clin.Invest.81 (4): 1012-9 (1988); Clezardin, P. etc., " Role of platelet membrane glycoproteins Ib/IX and IIb/IIIa; and ofplatelet alpha-granule proteins in platelet aggregation induced by humanosteosarcoma cells (effect of platelet α corpuscular protein in the inductive platelet aggregation of the effect of platelet membrane glycoprotein Ib/IX and IIb/IIIa and human osteosarcoma cell), " Cancer Res.53 (19): 4695-700 (1993).In addition, find a single polypeptide chain (60kd) with the closely-related hel cell film of GPIb surface expression, and corresponding to an incomplete or unusual O glycosylation GPIb α subunit.Kieffer, N. etc., " Expression of platelet glycoprotein Ibalpha in HEL cells (expression of platelet glycoprotein Ib α in the hel cell), " J.Biol.Chem.261 (34): 15854-62 (1986).
The GPIb complex
Each step all needs the existence of platelet surface receptor in hemostasis.A kind of receptor important in hemostasis is glycoprotein ibalpha-IX complex (being also referred to as CD42).This receptor mediation platelet is by (initial adhering to) blood vessel wall to the injury of adhering in conjunction with interior subcutaneous Feng Wei Lebulandeshi factor (vWF).It also plays crucial effect in the important platelet function in other two hemostasis: (a) by the inductive platelet aggregation of the high shear in stricture of artery district with (b) by the platelet activation of low concentration thrombin induction.
The GPIb-IX complex is one of main component of platelet plasma membrane outer surface.The GPIb-IX complex comprises three kinds and strides GPIb 130kDa α-chain and 25kDa beta chain and the non-covalent associating GPIX (22kDa) that membrane polypeptides-disulfide bond connects.For the effective function of cell surface expression and CD42 complex, all these 4 units all are present on the platelet membrane with equimolar amounts, show these 3 subunits correctly be assembled into complex be on plasma membrane, express fully needed.GPIb α-chain contains 3 particular structure territories: (1) is rich in the spherical N-terminal peptide domain of the flanking sequence of leucine repetitive sequence and cysteine bonding; (2) the huge glycopeptide domain of high glycosylation mucin sample contains GPIb β and the relevant C-terminal of the film district of striding film sequence and kytoplasm sequence disulphide bridges with (3).
Several evidences show that the thrombin binding structural domain of vWF and GPIb-IX complex is positioned at about 300 the amino acid whose spherical districts of the amino terminal that comprises GPIb α.Human blood platelets GPIb-IX complex is the key membrane receptor of mediation platelet function and reactive two aspects.Can make thrombocyte adhesiveness to the blood vessel of damaged by subcutaneous bonded vWF in the GPIb identification.In addition, vWF and GPIb α combine also induced platelet activation, and this may relate to the cytoplasmic structure territory of GPIb-IX and the interaction of cytoskeleton or phospholipase A2.And GPIb α contains the high-affinity binding site of α thrombin, and it promotes hematoblastic activation by a kind of mechanism of still knowing little about it so far.
The N-terminal globular domain of GPIb α contains amino bunch of negative charge.Several evidences show that in the transfection CHO cell of expressing the GPIb-IX complex and in platelet GPIb α, these three tyrosine residues are included in this domain (Tyr-276, Tyr-278 and Tyr-279) through oversulfated.
The protein sulphation
The protein sulphation is ubiquitous post translational modification, comprise vitriolic enzymatic covalently bound to or sugared side chain or polypeptide main chain on.This modification occurs in the trans-Golgi compartment, therefore only influences the protein that passes this compartment.Such protein comprises the extracellular region of secretory protein, grain targeting proteins and plasmalemma protein.Tyrosine is the Sulfated amino acid residue of present known process.J.W.Kehoe etc., Chemistry and Biol 7:R57-R61 (2000).Other aminoacid for example threonine perhaps also can be through oversulfated, especially in diseased cells.
Found that numerous protein is that tyrosine is Sulfated, still 3 of existence or a plurality of sulphation tyrosine are uncommon in the single polypeptide chain, as finding on GPIb.GPIb α (CD42) the mediation platelet of being expressed by platelet and megalokaryocyte is attached to interior subcutaneously and roll before subcutaneous interior by combine with vWF, also has many negative charges at its N-terminal domain.Think that such peracidity high-hydrophilic environment is the sulphation prerequisite, because the acid amino residue of tyrosyl albumen sulfotransferase specific recognition and make near the acid amino residue tyrosine sulphation.J.R.Bundgaard etc., JBC 272:21700-21705 (1997).The complete sulphation of the acidic region of GPIb α produces one and has the highdensity district of negative charge-in 19 aminoacid sequence sections 13 negative charges are arranged, and makes it become candidate position with other protein electrostatic interaction.
Select albumen and PSGL-1
It is adhesion molecule family that P, E and L select albumen, but its function especially mediated leucocytes on blood vessel endothelium before roll.P selects albumen to be housed in the hematoblastic granule, and is transported to particle surface after through thrombin, histamine, phorbol ester or the activation of other stimulation molecule.P selects albumen also can express on the activation endotheliocyte.E selects albumen to express on endotheliocyte, and L selects albumen to express on neutrophil cell, mononuclear cell, T cell and B cell.
PSGL-1 (being also referred to as CD162) is that P selects albumen, E to select albumen and L to select proteic mucin glycoprotein ligand.PSGL-1 is the homodimer with PACE (basic amino acid invertase in pairs) cleavage site that disulfide bond connects.PSGL-1 also has three potential tyrosine sulphation sites, and ten aggressiveness that described site is right after about 15 proline rich, serine and threonine repeat.The outer part of the born of the same parents of PSGL-1 contains three N linked glycosylation sites and numerous sialylated, fucosylation O connection oligosaccharide branches.K.L.Moore etc., JBC118:445-456 (1992).Most of N polysaccharide sites and many O polysaccharide site are occupied.The structure of the O polysaccharide of people HL-60 cell PSGL-1 is determined.The subgroup of these O polysaccharide is in conjunction with selecting required core-2, the sialylated and fucosylation structure of albumen.The tyrosine sulphation in the amino terminal district of PSGL-1 also is to select albumen and L to select albumen necessary in conjunction with P.In addition, there is the perhaps N-terminal propetide of translation back cutting.
PSGL-1 has 361 residues in the HL60 cell, comprise the extracellular region of 267 residues, the intracellular region of striding film district and 69 residues of 25 residues.The sequence of coding PSGL-1 is in an exon, so alternative splicing should be impossible.Yet, in the HL60 cell and the PSGL-1 in the most cells system have 15 of 10 residue consensus sequences in extracellular region, existing and repeat continuously, but this sequence is at polymorphonuclear leukocyte, mononuclear cell with comprise in other several cell lines of most of natural leukocyte having 14 and 16 repetitions.PSGL-1 forms the homodimer that disulfide bond connects at cell surface.V.Afshar-Kharghan etc., Blood 97:3306-3312 (2001).
PSGL-1 is expressed as dimer on neutrophil cell, the existing 250kDa of apparent molecular weight has 160kDa again, yet on HL60, described dimeric forms is～220kDa.When analyzing under reducing condition, only half is reduced each subunit.The difference of molecular mass may be owing to repeated to exist due to the polymorphism of generation by ten aggressiveness of different numbers in the described molecule.Leukocyte Typing the 5th edition, T.Kishimoto etc. write, (1997).
PSGL-1 for example expresses on neutrophil cell, mononuclear cell, leukocyte, B cell subsets and all the T cells at most of blood leucocytes, and the mediation neutrophil cell selects albumen to go forward to roll at P.Leukocyte Typing the 5th edition, T.Kishimoto etc. write, (1997).PSGL-1 also can be by selecting protein bound interaction to mediate neutrophil cell-neutrophil cell with L, thus transmitting inflammation.Snapp etc., Blood91 (1): 154-64 (1998).
The PSGL-1 mediated leucocytes activation on endothelium, activated blood platelet and other leukocyte and the inflammation part before roll.
But the anti-people PGSL-1 monoclonal antibody KPL1 that a kind of commercialization obtains has produced and has shown and can suppress between PGSL-1 and the P selection albumen and the interaction between PGSL-1 and the L selection albumen.The KPL1 epitope mapping is to the tyrosine sulphation consensus motif (YEYLDYD) of PGSL-1.KPL1 only discerns this special epi-position, and not with for example B-CLL cell, AML cell, transitional cell, multiple myeloma cells etc. are gone up the sulphation epi-position cross reaction that exists on other cell.
It is important rolling before the leukocyte in inflammation, and the adhesion of interaction partners leukocyte on blood vessel wall between P selection albumen (expressed on platelet by the activation endothelium, can be fixed on the injury) and PSGL-1 is helpful with before rolling.Ramachandran etc., PNAS98 (18): 10166-71 (2001); Afshar-Kharghan etc., Blood 97 (10): 3306-7 (2001).
Rolling before the cell also is important in transfer, and believes that the P on the endotheliocyte selects albumen and E to select the protein binding transitional cell, enters into surrounding tissue thereby impel to ooze from blood flow at home and abroad.
Platelet also participates in transfer process; When the metastatic carcinoma cell enters blood flow, can form the many cells complex of being formed by the leukocyte of tumor cell by platelet and bag.These complex can be called micro-embolization, help tumor cell to escape immune system.Need express P by platelet by the tumor cell of platelet bag quilt and select albumen.
Can suppress tumor cell-hematoblastic interaction with heparin (P selects albumen and L to select proteic inhibitor) treatment.Spend except O sialoglycoprotein enzyme pretreatment tumor cell sialylated, fucosylation mucin part, also suppress the formation of tumor cell-platelet complex.The interior experiment of body shows that each can produce the more mononuclear cell relevant with circulating tumor cell in these processing, but prompting minimizing platelet combination enhance immunity cell is near the circulation tumor cell.Varki and Varki, Braz.J.Biol.Res.34 (6): 711-7 (2001).
PSGL-1 and GPIb share and have the structural similarity of mucin sample, high glycosylation ligand binding domain.Afshar-Kharghan etc., Blood 97 (10): 3306-7 (2001).
PSGL-1 is present at all leukocyte and is on neutrophil cell, mononuclear cell, lymphocyte, activation periphery T cell, granulocyte, eosinophilic granulocyte, the platelet and is present on the positive stem cell of some CD34 and some the B cell subsets.P selects albumen selective expression on activated blood platelet and endotheliocyte.P select the interaction between albumen and the PSGL-1 promote leukocyte on blood vessel wall before roll, and leukocyte causes various pathologic inflammation in the unusual accumulation of vascular site.It is important with combining of PSGL-1 that the Sulfated stereospecificity contribution of the last indivedual tyrosine of PSGL-1 is selected albumen to P.Electric charge is important also for combination: and minimizing NaCl (reducing to 50mM from 150mM) enhancing combination (Kd～75nM).The last tyrosine sulphation of PSGL-1 can strengthen PSGL-1 and adhere to P and select on the albumen, but is not that PSGL-1 adheres to P and selects on the albumen necessary after all.The support of PSGL-1 tyrosine sulphation is more before rolled adhesion with various shear rates, supports again to roll adhesion before the much higher shear rate.(Rodgers SD etc., Biophys is (2001) J.81:2001-9).
Fibrinogen
Normal human fibrinogen has two kinds of forms: the main variant of fibrinogen γ and fibrinogen γ ' minor variations, wherein each all is present in the normal individual.Normal fibrinogen is more abundant form (accounts for the fibrinogen that exists in the body 90%), is made up of two identical 55kDa α chains, two identical 95kDa β chain 49.5kDa γ chains identical with two.Normal fibrinogen variant is the form (account for the fibrinogen that exists in the body 10%) of less abundance, is made up of two identical 55kDa α chains, two identical 95kDa β chains, a 49.5kDa γ chain and a 50.5kDa γ ' chain.γ chain and γ ' chain are all by same gene code, and this gene has the alternative splicing that exists at 3 ' end.Normal γ chain is made up of amino acid/11-411.Normal γ ' chain variants is made up of 427 aminoacid: amino acid/11-the 407th, and the same with those aminoacid in the normal γ chain, and aminoacid 408-427 is VRPEHPAETEYDSLYPEDDL.This district is occupied by the thrombin molecule usually.
When fibrinogen existed at ionized calcium, the role transformation by thrombin became fibrin, causes blood coagulation.Fibrin also is thrombosis composition and acute inflammation exudate.
Platelet and in cell-cell interaction; cell-matrix interacts; platelet-platelet interacts; platelet-cell interaction; platelet-matrix phase mutual effect; roll before the cell and adhere and stop blooding in the molecule (fibrinogen for example that plays an important role; GPIb; select albumen and PSGL-1) also in pathogenic disease or for example unusual or pathogenic inflammation of morbid state; unusual or pathogenic immunoreation; autoimmune response; neoplasm metastasis; unusual or pathogenic adhesion; play an important role in thrombosis and/or restenosis and the unusual or pathogenic gathering.Therefore, can be used for diagnosing with the antibody of platelet and these molecule cross reactions and treat in comprise the disease and the obstacle of these and other pathogenic disease.
An object of the present invention is to provide the compositions of antibody or its fragment and medicine, described medicine comprises anticarcinogen, antimetastatic agents, antileukemia, disease-resistant medicine, antisticking medicine, antithrombotic drug, anti-restenosis, anti-autoimmune medicine, the anti-medicines such as medicine, antimicrobial drug, antiviral agents or anti-inflammatory agent of assembling.
Another object of the present invention provides utilizes medicine and antibody or its fragment to treat the method for various diseases, described disease comprise those with cell before roll relevant disease; Inflammation; Autoimmune disease; Neoplasm metastasis; The growth of tumor cell and/or propagation; The mortality rate of tumor cell; Leukaemia's growth and/or propagation; Leukaemia's mortality rate; The sensitivity of diseased cells enantiopathy medicine damage; Tumor cell is to the sensitivity of antineoplastic agent damage; The leukaemia is to the sensitivity of antileukemia damage; Leukemia cell number in tumor or cancer patient's interior tumor cell number and the leukaemic's body.
A further object of the present invention provides utilizes medicine and antibody or its fragment to carry out the method for therapeutic treatment, the amount of the clinical effective of this medicine that wherein said medicine and/or antibody or the content of its fragment in compositions were found when being lower than described medicine and giving separately usually.
This paper provides these purposes of the present invention and other purpose.
Summary of the invention
The invention provides and contain the segmental compositions of medicine and antibody or its.The present composition is that such compositions is so that described medicine can be compound with described antibody or its fragment; Described medicine can combine with described antibody or its fragment; Perhaps described medicine can be puted together with antibody or its fragment.The antibody of the present composition or its fragment can exist with one or more antibody or segmental complex or aggregation.
In the present composition, described medicine and/or described antibody or its fragment can be subclinical amount.This subclinical quantity not sufficient is with the sensitivity of effective change sensitivity to medicine, particularly diseased cells.Described medicine can be anticarcinogen, antimetastatic agents, antileukemia, disease-resistant medicine, antisticking medicine, antithrombotic drug, anti-restenosis, anti-autoimmune medicine, anti-medicine, antimicrobial drug, antiviral agents or the anti-inflammatory agent assembled.Preferred described medicine is the anthracycline or derivatives thereof, can be doxorubicin, daunorubicin, idarubicin, morpholine doxorubicin, morpholine daunorubicin, methoxy morpholine doxorubicin or their derivant or combination.Most preferably described medicine is the doxorubicin or derivatives thereof.
In addition, the subclinical quantity not sufficient of described medicine roll before with effective inhibition cell, inflammation, autoimmune disease, thrombosis, restenosis, neoplasm metastasis or tumor cell or leukaemia's survival, growth and/or propagation.In addition, the subclinical quantity not sufficient of described medicine is with the increase of effective inhibition tumor patient interior tumor cell number or be not enough to effectively suppress the increase of leukemia cell number in leukaemic's body.Equally, the subclinical quantity not sufficient of described medicine is with the intravital tumor cell number of effective minimizing tumor patient or to be not enough to effectively reduce the leukaemic intravital from the disorders of blood cell number.In addition, the subclinical quantity not sufficient of described medicine with effective increase tumor cell or leukaemia's mortality rate, be not enough to effectively to increase tumor cell to the sensitivity of anticarcinogen damage or be not enough to effectively increase the sensitivity of leukaemia to the antileukemia damage.At last, the subclinical quantity not sufficient of described medicine forms, assembles or adhesion with effective inhibition cell-cell, cell-matrix, platelet-substrate, platelet-platelet and/or cell-platelet complex.
The antibody of the present composition or its fragment can have the binding ability of the scFv antibody fragment of SEQ ID NO:1, SEQ IDNO:2 or SEQ ID NO:3.In addition, described antibody or its fragment can have the binding ability of peptide or polypeptide, and wherein said peptide or polypeptide have first hypervariable region of SEQ ID NO:4.This peptide or polypeptide can also have second hypervariable region of SEQ ID NO:5 and/or the 3rd hypervariable region of SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.Described antibody or its fragment can be scFv or Fab fragment.
The present composition also can contain medicine and antibody or its fragment, wherein said antibody or its fragment are in conjunction with length about 3 peptide or polypeptide epitopes to about 126 amino acid residues, and described peptide or polypeptide epitope have at least 2 acidic amino acids and at least one sulphation tyrosine residue.In addition, compositions of the present invention can contain medicine and antibody or its fragment, wherein said antibody or its fragment are in conjunction with at least two kinds of different molecules, and described molecule is selected from PSGL-1, fibrinogen γ ', GP1b α, heparin, chamber Dan Baijutang (lumican), complement chemical compound 4 (CC4), interalpha inhibitor and thrombinogen.Same in addition, compositions of the present invention can contain medicine and antibody or its fragment, wherein said antibody or its fragment are in conjunction with at least two kinds of different molecules with in conjunction with at least a cell type, described molecule is selected from PSGL-1, fibrinogen γ ', GP1b α, heparin, chamber Dan Baijutang, complement chemical compound 4 (CC4), interalpha inhibitor and thrombinogen, and described cell type is selected from B cell leukemia cell, B-CLL cell, AML cell, multiple myeloma cells and transitional cell.In addition, compositions of the present invention can contain medicine and antibody or its fragment, wherein said antibody or its fragment and two or more epi-position cross reaction, each epi-position comprise one or more sulphation tyrosine residues and two or more acidic amino acids at least one cluster.
In compositions of the present invention, described antibody or its fragment can with vehicle or carrier coupling or compound or combine, described vehicle or carrier are with more than a kind of drug coupling or associating or combine.This vehicle or carrier can be selected from glucosan, lipophilic polymer, hydrophilic polymer, HPMA and liposome.Described vehicle or carrier be the liposome of doxorubicin modification preferably.Perhaps, preferably Polyethylene Glycol (PEG) or glucosan of described vehicle or carrier.
The present invention also provides the method that compositions of the present invention is had the patient who needs.For example, by any compositions of the present invention there being the patient who needs, the present invention also provides the method for alleviating disease effector, prevent disease, treatment disease or suppressing disease process.The invention provides and a kind ofly suppress to roll before the cell, inflammation, autoimmune disease, thrombosis, restenosis, transfer, tumor cell existence, growth and/or propagation, leukaemia's growth and/or propagation, increase the intravital tumor cell number of tumor patient or increase the method for the intravital leukaemia's number of leukaemic.In addition, the invention provides mortality rate, the mortality rate that increases the leukaemia, the sensitivity that increases the damage of diseased cells enantiopathy medicine that increase tumor cell, increase tumor cell to the sensitivity of anticarcinogen damage or increase the method for leukaemia to the sensitivity of anticarcinogen damage.In addition, the invention provides the method that reduces the intravital tumor cell number of tumor patient, reduces the intravital leukaemia's number of leukaemic.At last, the invention provides the method that inhibition cell-cell, cell-matrix, platelet-substrate, platelet-platelet and/or cell-platelet complex forms, assembles or adhere.
The present invention also provides a kind of method of therapeutic treatment, and described method is by having patient's (i) antibody of needing or its fragment and (ii) medicine, and wherein one or both described antibody or its fragment and/or described medicine give with subclinical amount.In the method, described medicine and antibody or its fragment can put together, compound or unite and give.In addition, described medicine can give separately, gives before or after antibody or its fragment giving, and perhaps gives by any other order, and vice versa.
Definition
Antibody (Ab) or immunoglobulin (IgG) are the protein molecules of conjugated antigen.They are made up of four polypeptide chains that link together by disulfide bond (2 heavy chain and 2 light chains) unit.Every chain all has constant region and variable region.Heavy chain component according to them can be divided into five classes: IgG, IgM, IgA, IgD and IgE.The IgG class comprises several subclass, includes but not limited to IgG 1, IgG 2, IgG 3And IgG 4Immunoglobulin is produced in vivo by bone-marrow-derived lymphocyte, and discerns specific exogenous antigen determinant and impel this antigenic removing.
Antibody can prepare and use in a variety of forms, comprises antibody complex.As used herein term " a kind of antibody complex " or " multiple antibody complex " be used in reference to one or more antibody and another kind of antibody or with an antibody fragment or many complex that antibody fragment forms, the perhaps complex of two or many antibody fragments.The example of antibody fragment comprises Fv, Fab, F (ab ') 2, F (ab '), Fc and Fd fragment.
As in the description of the present invention and claim in used, Fv is defined as the molecule of being made up of the variable region of light chain of the variable region of heavy chain of people's antibody and people's antibody, they can be identical or different, and wherein variable region of heavy chain connection, connection, fusion or covalently bound to or associate to variable region of light chain.Fv can be the stable Fv (dsFV) of strand Fv (scFV) or disulfide bond.ScFV is made up of each the bar heavy chain of antibody and the variable region of light chain, is connected by flexible amino acid polypeptide spacer or joint.This joint can be ramose or unbranched.Preferred joint is a 0-15 amino acid residue, and most preferably joint is (Gly 4Ser) 3
The Fv molecule itself comprises first chain and second chain, and every chain comprises first, second and the 3rd hypervariable region.Hypermutation ring in variable region of light chain and the variable region of heavy chain is called as complementary determining region (CDR).In every heavy chain and the light chain CDR1 district, CDR2 district and CDR3 district are arranged.Believe that these districts form antigen-binding site, and can modify to produce enhanced specifically in conjunction with active.The CDR3 district that is actually heavy chain that these districts are the most variable.It is the exposed region of Ig molecule that CDR3 infers in the district, and as shown here and provide main position of being responsible for selectivity and/or observed specificity binding characteristic.
The fragment of Fv molecule is defined as less than original Fv but still keeps the selectivity of original Fv and/or any molecule of specificity binding characteristic.This segmental example includes but not limited to (1) small-sized antibody (minibody), the fragment that only comprises the Fv heavy chain, (2) miniantibody (microbody), the small fragment (PCT application number PCT/IL99/00581) that comprises antibody heavy chain variable region, (3) comprise the similar antibody of light chain segments and the analogue that (4) comprise the functional unit of variable region of light chain.
Term used herein " Fab fragment " is the monovalent antigen binding fragment of immunoglobulin.The Fab fragment is made up of the part of light chain and heavy chain.
F (an ') 2Fragment is the bivalence Fab by the immunoglobulin of pepsin digestion acquisition.It contains the part of two light chains and two heavy chains.
The Fc fragment is the non-antigen-binding portion thereof of immunoglobulin.It comprises the carboxyl terminal part and the Fc receptor binding site of heavy chain.
The Fd fragment is the variable region and first constant region of heavy chain immunoglobulin.
Polyclonal antibody is the product of immunne response, and is formed by many different B-lymphocytes.Monoclonal antibody is derived from individual cells.
Be applicable to that polypeptide and defined in the present invention box are meant one section given continuous amino acid sequence, it is as framework and think a unit, and can be by a unit operation.Aminoacid can be at one end or the two ends displacement, is inserted, removes or connect.Equally, the aminoacid sequence section can be at one end or the two ends displacement, is inserted, removes or connect.
Term used herein " epi-position " is meant with antibody, antibody fragment, antibody complex or comprises complex or the interactional antigenic determinant of TXi Baoshouti or the antigen position of its binding fragment.Term epi-position and term part, domain and land are used interchangeably in this article.
Selectivity is defined as in this article that targeted molecular is selected and in conjunction with the ability of a kind of cell type or cell state from may the mixture to the various cell types of described targeted molecular specificity or cell state, all cell type or cell state.
Term used herein " affinity " is the measured value (association constant) of bond strength between receptor (a for example binding site on the antibody) and the part (for example antigenic determinant).The intensity of the noncovalent interaction sum between antigen-binding site on the antibody and the epi-position is the affinity of this antibody to this epi-position.Hang down weak conjugated antigen of affinity antibody and trend and dissociate easily, however more closely conjugated antigen and the more permanent combination of maintenance of high-affinity antibody.Term " affinity " is different from affinity, because the former reflects the price of antigen-antibody interaction.
The specificity of antibody-AI: though antigen-antibody reaction is specific, in some cases by a kind of antibody of antigen induced can with other irrelevant antigenic cross-reaction.When if if two different antigen is shared homology or similar structure, epi-position or its anchorage zone or a kind of epitope specificity antibodies and had the irrelevant epi-position of analog structure conformation or chemical property, this cross reaction can take 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.
The conserved amino acid replacement is defined as by changing peptide, polypeptide or protein or its segmental one or two aminoacid and changes amino acid whose composition.Described replacement normally has the aminoacid of similar characteristic (for example acidity, alkalescence, aromatics, size, positive charge or negative charge, polarity, nonpolar), can not change peptide, polypeptide or protein characteristic (for example electric charge, IEF, affinity, affinity, conformation, dissolubility) or activity aspect main so replace substantially.May realize that the typical case that this conserved amino acid replaces replaces and can take place in 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)
Conserved amino acid replaces also and can mainly be responsible in the hypervariable region of molecular selectivity and/or specificity binding characteristic in adjacency, and the other parts of molecule, for example carries out in the weight chain variable box.In addition, can form antibody, bifunctional antibody (diabody) (dimer), three function antibodies (triabody) (trimer) and/or four function antibodies (tetrabody) (tetramer) of full size or form small-sized antibody or miniantibody is finished modification by rebuilding molecule.
Phasmid is defined as the phage particle that carries plasmid DNA.Phasmid is the plasmid vector that design comprises the ml3 of filobactivirus replication origin such as fd.Because it carries plasmid DNA, so this phase granule does not have enough spaces to comprise the complementary series of complete phage genome.The composition that lacks in described phage genome is a bacteriophage particles packing information necessary.So,, be necessary to cultivate required phage particle with having replenished the described helper phage strain that lacks packaged information in order to breed described phage.
Promoter is that DNA goes up RNA polymerase combination and the initial district that transcribes.
Phage display library (being also referred to as phage display peptide/antibody library, phage library or peptide/antibody library) comprises a big phage colony (common 10 8-10 9), each phage particle is showed a different peptide or peptide sequence.These peptides or polypeptide fragment can be built into length variable.Peptide that is demonstrated or polypeptide can be derived from but be not limited to human antibody heavy chain or light chain.
Pharmaceutical composition is meant the preparation that comprises peptide of the present invention or polypeptide and pharmaceutically acceptable carrier, excipient or diluent.
Medicine is meant the medicine that can be used for prophylactic treatment or diagnosis mammal (including but not limited to people, cattle, horse, pig, Mus, dog, cat or arbitrary other homoiothermic animal).Described medicine is selected from radiosiotope, toxin, oligonucleotide, recombiant protein, antibody fragment and anticarcinogen.The example of this class medicine includes but not limited to antiviral agents, comprises acyclovir, ganciclovir and zidovudine; Antithrombotic drug/anti-restenosis comprises cilostazol, dalteparin sodium, Reviparin Sodium and aspirin; Anti-inflammatory agent comprises zaltoprofen, pranoprofen, drogelor, acetyl salicylic acid 17, diclofenac, ibuprofen, dexibuprofen, sulindac, naproxen, ammonia tolmetin, celecoxib, indomethacin, rofecoxib, nimesulide; Anti-autoimmune medicine comprises leflunomide, denileukin diftitox, subreum, WinRho SDF, defibrotide and cyclophosphamide; Antisticking medicine/anti-the medicine of assembling comprises limaprost, cloricromen and hyaluronic acid.
Antileukemia is the medicine with anti-leukocythemia liveness.For example, antileukemia comprises that the medicine that suppresses or stop leukaemia or the growth of immature Preleukemia cell, the medicine that kills leukaemia or Preleukemia cell, increase leukaemia or Preleukemia cell are to the medicine of the sensitivity of other anti-leukemia medicine and the medicine of inhibition leukaemia transfer.In the present invention, antileukemia also can be the medicine with anti-angiogenesis activity, forms thereby prevent, suppress, postpone or stop tumor vessel.
Can by analyzing gene under various conditions, at special time, in the various product amounts of organizing medium generation, the expression of gene pattern is studied.When the amount of gene outcome is higher than the product amount of finding, can think that then gene is " overexpression " in for example non-ill contrast of normal control.
Given cell can have the protein of given antibody combining site (or epi-position) at its surface expression, but this binding site can be with the form hidden (for example by steric hindrance or be closed, or the essential feature of shortage antibodies) be present in a kind of state of described cell, this phenomenon can be called the first phase (I phase).The I phase can be for example normal, healthy, non-disease state.When epi-position existed with the form of hiding, it can promptly not have antibodies to the given cell of this epi-position or I phase by given antibody recognition.Yet described epi-position can be by for example modifying through self, or because contiguous or relevant molecule is modified or because certain district remove sealing and expose through conformation change.The example of modifying comprises folding variation, post translational modification variation, phospholipid variation, sulphation variation, glycosylation variation etc.When cell enters different states, these modifications can take place, this phenomenon is called the second phase (II phase).The example of the second phase etc. comprises activation, breeds, transforms or is in malignant state.Through modifying, then can expose epi-position and antibody can in conjunction with.
Peptide mimics is to have identical functions effect or another entity for example active micromolecule, peptide, polypeptide, lipid, polysaccharide or its conjugate of antibody.
The accompanying drawing summary
Independent, the sequential or percentage survival of uniting the mice that gives to carry behind doxorubicin and the Y1 MOLT-4 tumor that Fig. 1 illustrates to the time (my god) curve chart.
Detailed Description Of The Invention
The present invention relates to comprise composition and the method for medicine and antibody or its fragment. This Bright composition can be to cause one or more antibody or its fragment different from one or more Medicine gathering, association, compound or in conjunction with or put together, merge or connect, for example medicine, Toxin and radio isotope optional together with pharmaceutically effectively carrier form have disease-resistant Medicine-the peptide complexes of activity and/or active anticancer, composition or conjugate. This compound, Composition or conjugate also can be used for diagnostic purpose. In addition, described medicine and antibody are described Can be subclinical amount in the composition. Subclinical amount refers to less than working as described medicine and/or antibody Usually the medicine of finding when giving separately and/or the amount of the clinical best effective dose of antibody. Subclinical Amount also can refer to less than usually find, cause medicine that known clinical response is essential and/or The amount of antibody amount. People will recognize that, when thing combined according to the invention and method administration, Subclinical amount does not refer to that described medicine and/or antibody are invalid clinically.
In a preferred embodiment, the subclinical amount of described medicine can be to be not enough to Effectively change the sensitiveness to the sensitiveness, particularly diseased cells of medicine. For example, described The subclinical amount of medicine can be not enough to roll before the establishment cell, inflammation, autoimmunity Disease, thrombosis, ISR, transfer or tumour cell or leukaemia's growth and/ Or propagation. In addition, the subclinical amount of described medicine can be to be not enough to the establishment tumour suffer from The increase of person's interior tumor cell number or be not enough to establishment leukaemic body words spoken by an actor from offstage blood The increase of sick cell number. The subclinical amount of described medicine also can be to be not enough to effectively reduce swell Knurl patient body inner tumour cell number or be not enough to effectively reduce leukaemia in leukaemic's body Cell number. In addition, the subclinical amount of described medicine can be that to be not enough to effectively to increase tumour thin Born of the same parents or leukaemia's the death rate is not enough to effectively to increase tumour cell anticarcinogen is damaged Sensitiveness, perhaps be not enough to effectively to increase quick to the antileukemia damage of leukaemia Perception. At last, the subclinical amount of described medicine can be not enough to establishment cell-cell, Cell-matrix, blood platelet-matrix, blood platelet-blood platelet and/or cell-blood platelet compound shape Become, assemble or adhesion.
The preferred present composition contain have in conjunction with SEQ ID NO:1, SEQ ID NO:2 or Antibody or its fragment of the scFv antibody fragment ability of SEQ ID NO:3. SEQ ID NO:1 The scFv antibody fragment be appointed as Y1, the scFv antibody fragment of SEQ ID NO:2 is Through being appointed as Y17, the scFv antibody fragment of SEQ ID NO:3 has been appointed as L32. Logical Crossing screening only has multifarious people's antibody phage library and can identify in heavy chain CDR3 district These antibody. With regard to people scFv Y1 and Y17 antibody, in order to identify in conjunction with hematoblastic Antibody can screen the immobilization human blood platelets. The L32 of screening leukemia cell is to select knowledge The specific antibody of other leukaemia surface determinant is not before wherein specific receptor is Known or sign. Profit uses the same method and can identify another antibody L31.
Before, be used for the antibody of the present composition at U. S. application the 10/032nd, 423 Number; The 10/032nd, No. 037; The 10/029th, No. 988; The 10/029th, No. 926; The 09/751st, 181 Number; With the 60/258th, No. 948 and international application no PCT/US01/49442 and Utilize identical phage library to identify among the PCT/US01/49440. In these applications In disclosed concrete antibody example comprise Y1 and Y17 antibody. Discovery is public in these applications The antibody of opening can be attached on the epi-position that exists on the hematopoietic cell protein specifically, these albumen The terminal tyrosine of the N of matter is sulphation and think that it participates in turning to of cell migration such as tumour Move.
The epi-position of Y1 antibody is positioned at glycocalyx albumen, in the subunit of a CD42 compound Amino acid 272 and 285, wherein the CD42 compound has the band that is produced by the sulphation group Negative electrical charge amino acid bunch, for Y1 and glycocalyx protein combination necessary. In addition, Y1 combination The N end of PSGL-1, PSGL-1 are the acceptors that E, L and P select albumen, comprise sulphur The tyrosine residue of electronegative amino acid bunch is followed in acidifying. Although Y1 antibody is in conjunction with several branches Son, for example the glycocalyx protein molecular on the blood platelet, fibrinogen γ ', human plasma complement chemical combination PSGL-1 molecule on thing 4 and the KG-1 cell, it is to being derived from AML or multiple marrow Knurl (MM) patient's leukocytic compatibility of former generation wants high several with respect to previously mentioned epi-position The order of magnitude.
In addition, L32 and L31 antibody are disclosed in the U. S. application of application on July 1st, 2002 60/__, _ _ number in, denomination of invention is " L32 Antibodies and Uses Thereof (L32 Antibody and application thereof) ". Although L32 is with the compatibility combination white blood more about 5 times than the Y1 height Sick cell, but L32 antibody and disclosed antibody all can be in conjunction with white blood in the Y1/Y17 application Sick cell. Although L32 and Y1/Y17 antibody are all separated from common kind system (DP32), And as if L32 is in conjunction with the sulphation epi-position identical with Y1/Y17, but L32 is not in conjunction with blood Platelet, and do not affect platelet aggregation.
The previous sulphation epi-position in conjunction with preferred antibody of the present invention of identifying is characterised in that existence The sulphation part, for example sulphation tyrosine residue or sulfated sugar the part or lipid part, Preferably in two or more acidic amino acids bunch, part and acceptor find described poly-Bunch different processes such as inflammation, immune response, infection, autoimmune response, transfer, Roll before adhesion, thrombosis and/or ISR, the cell and assemble in play an important role. This Epi-position also is present in diseased cells for example B cell leukemia cell, B-CLL cell, AML On cell, multiple myeloma cells and the transitional cell. These epi-positions are controlled these processes Treating mediation and diagnosing operation is useful target.
The antibody of the preferred present composition is in conjunction with the different molecular or the epi-position that participate in inflammation, example Such as PSGL-1, fibrinogen γ ', GP1b, heparin, LUM, complement compound 4 (CC4), interalpha inhibitor and factor. Also preferred antibody is in conjunction with being present at least A kind of epi-position that participates on inflammation or the tumorigenic cell type, comprise the B-CLL cell, T-ALL cell, AML cell, B-leukaemia, multiple myeloma cells and transfer Cell. More preferably the antibody of the present composition is in conjunction with lipid, sugar, peptide, glycolipid, sugared egg In vain, the epi-position on lipoprotein and/or the lipopolysaccharide molecule. Such epi-position preferably has at least one Individual sulphation part. Perhaps, but also preferred described antibody intersects with a plurality of epi-positions with two Reaction, each epi-position have one or more sulphation tyrosine residues and at least one contains two Individual or a plurality of acidic amino acids bunch, the example is PSGL-1.
What participate in the formation antigen-binding site is the hypervariable region of antibody of the present invention. Described antigen knot Close the epi-position complementary structure of being combined with antibody at the position; Therefore these are called as complementarity in conjunction with the position Determining area (CDR). Every light chain and heavy chain at antibody have three CDR (CDR1, CDR2 And CDR3), each is positioned at and connects VHDistrict and VLOn the ring of the β chain in district. In these districts The most variable is the CDR3 district of heavy chain. Think that the CDR3 district is the district that the Ig molecule exposes most, In the selective and/or specific binding feature of the decision of observing such as having of providing of this paper Heart effect.
In a preferred embodiment of the present composition, described antibody or its fragment tool First hypervariable region (CDR3) that SEQ ID NO:4 is arranged. In addition, perhaps, described antibody or its Fragment has second hypervariable region (CDR2) of SEQ ID NO:5. In addition, perhaps, described anti-Body or its fragment have of SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8 Three hypervariable regions (CDR1).
According to the present invention, CDR also can be inserted in the box, produces antibody. As be applicable to many Peptide and the box as defining among the present invention refer to the given sequence as the continuous amino acid of framework Be thought of as a unit and as a unit operation. Amino acid can be at one end or the two ends quilt Displacement, insertion, removal or connection. Equally, the amino acid sequence section can be at one end or two ends Replaced, insert, remove or connect. The amino acid sequence of described box can be solid on the surface Fixed, and replaced, insert or the sequence that connects can be hypermutation. Described box can wrap Contain several domains, each domain comprises the crucial function of final construct. The box of a specific embodiments of the present invention from N terminal comprise framework region 1 (FR1), CDR1, framework region 2 (FR2), CDR2, framework region 3 (FR3) and framework region 4 (FR4). In one embodiment of the invention, the unique district of displacement is possible in described box. For example, the CDR2 of described box and CDR1 hypervariable region can be replaced by non-conserved amino acid, Perhaps preferred conserved amino acid replaces displacement or modifies.
For all describe in detail herein≤amino acid sequence (example of 25 amino acid residues Such as CDR district, CDR flanking region), people know and recognize, as of the present invention another Embodiment, these amino acid sequences are included in one or two 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor scope, Preferred described replacement is that conserved amino acid replaces. For all describe in detail herein＞25 The amino acid sequence of amino acid residue, people know and recognize, as one of the present invention Embodiment, these amino acid sequences are included in an amino acid sequence to be had original series Have＞90% sequence similarity scope interior (Altschul etc., Nucleic Acids Res.25:3389-402 (1997)). Amino acid similar or homology be defined as the performance similar characteristic for example acid, alkalescence, Aromatics, size, positive charge or negative electrical charge, polarity, nonpolar different aminoacids.
The percentage of amino acid similarity or homology or sequence similarity can be by relatively two Different peptides or the amino acid sequence of polypeptide are determined. Antibody sequence can be determined by dna sequencing. Usually one of multiple computer program that can be by utilizing for this purpose design advances two sequences The row comparison, and compare each locational amino acid residue. Determine that then amino acid is same Property or homology. Use then algorithm and measure amino acid similarity percentage. Owing to extremely increase The sensitivity of sensitive relations between strong detection of peptides, polypeptide or protein molecule, usually preferably than Than amino acid sequence. Protein relatively can be considered the existence that conserved amino acid replaces, if Different aminoacids has similar physics and/or chemical characteristic, and mispairing also may produce positive score value (Altschul etc., (1997), the same).
In one embodiment of the invention, three hypervariable regions of every light chain and heavy chain can With in two interchains and the chain in three hypermutation positions and/or interchain exchange.
The invention provides the peptide with antibody or its fragment, its construct or fragment construct Or polypeptide. According to the present invention, antibody comprises IgG, IgA, IgD, IgE or IgM antibody. The IgG class comprises several subclass, comprises IgG1、IgG 2、IgG 3And IgG4。
Antibody can provide in a variety of forms, for example fragment, compound and polymer. According to The present invention, antibody fragment comprises Fv, scFv, dsFv, Fab, Fab2With the Fd molecule. Little antibody fragment for example various Fv fragments and various Fab fragment is also included within term " fragment " In, if they keep original antibody or than large fragment in conjunction with feature. The reality of these fragments Regular meeting is (1) small-sized antibody, only comprises the Fv heavy chain fragment, (2) miniantibody, and it is heavy to comprise antibody The fraction unit of chain variable region (international application no PCT/IL99/00581), (3) have the light chain sheet The similar antibody of section; (4) has the similar antibody of the functional unit of variable region of light chain. Make up Body comprises for example polymer such as bifunctional antibody, three function antibodies and four function antibodies. Phrase " antibody or its fragment or have antibody or the compound of its fragment " and " antibody or sheet The section " expection comprise these all molecules with and derivative and homologue, analogies and change Body is unless the knowledge of based on the context and/or this area describes in detail in addition or points out in addition.
Having set up the scFv infiltration organizes and removes from blood quickly than the antibody of full size Method because their in size littler (Adams etc., Br.J.Cancer 77:1405-12 (1988); Hudson, Curr.Opin.Immunol.11 (5): 548-557 (1999); Wu Deng, Tumor Targeting 4:47 (1999)). Thereby scFv often is applied to diagnostics, Comprise for example tumor imaging of radioactive label, in order to from health, remove quickly described radiation The property mark. Many cancer target scFv polymers face body internal stability and effect process in the recent period Assessment before the bed (Adams etc., (1988) are referring to above; Wu (1999) is referring to above).
Usually, design has VHThe C end in district is connected to V by peptide linkerLThe N end The scFv monomer of end residue. Can choose wantonly and use rightabout: VLThe C end in district is connected to V by peptide linkerHN terminal residue (Power etc., J.Immmun.Meth.242:193-204 (2000)). Described peptide linker length is generally about ten five amino acids. When joint reduces to About three during to seven amino acid, and then scFv can not be folded in the functional Fv district and change into and Two scFv associate and form bifunctional antibody. According to joint length, composition and Fv district direction, more The length that further reduces described joint forces scFv to associate into three to being less than three amino acid Aggressiveness or the tetramer (Powers (2000), referring to above).
Recently, multivalent antibody fragment for example scFv dimer, tripolymer and the tetramer have been found Usually can provide higher compatibility to be attached on the target than its parental antibody. This higher parent Provide potential advantage with property, comprised and improve the pharmacokinetics that cancer target is used. In addition Outward, the P selection albumen and its part PSGL-1 that participate in leukocytic adhesion and before roll in research In, scientists is inferred, expresses the cell of PSGL-1 dimeric forms, because more this High binding affinity and set up and rolled adhesion before more stable. Before adhering to, these roll speed Fluctuation (Ramachandran etc., PNAS, 98 are still less resisted and showed in the aspect more fully (18): 10166-71 (2001)).
The higher binding affinity of these multivalence forms may be conducive to diagnosis and therapeutic scheme. For example, can use scFv as the blocking agent in conjunction with the target acceptor, and therefore blocking-up " natural " The combination of part. In this case, what people were required is, between scFv and described acceptor Have the association than high-affinity, with the possibility that minimizing is dissociated, this dissociate may so that Native ligand undesirably is attached on the target. In addition, when described target acceptor participate in adhesion and Before when rolling, or when cell that described target acceptor is present in high flow region is for example on the blood platelet, This high-affinity may be useful.
In case screen and/or develop antibody, fragment or the construct with required binding ability, Then produce construct and the fragment of the feature that keeps original antibody, just in time utilizing this paper to provide In those skilled in the art's limit of power that instructs. For example, can prepare the required original sieve of maintenance Complete antibody molecule, Fv fragment, the Fab of antibody, fragment or construct characteristic choosing or exploitation Fragment, Fab2Fragment, dimer, tripolymer and other construct.
If need substituted amino acid, still still keep the feature of antibody or fragment, then preparation Conserved amino acid replaces just in time in the art technology scope. Also can antagonist or fragment advance The various modifications of row, for example with medicament diagnosticum is compound or in conjunction with or put together and do not change them In conjunction with feature. Also can antagonist or fragment to carry out other for example those generations more stable Antibody or the various modifications of fragment and do not change their specificity. For example, can intend Peptide (peptoid) modifies, partly intends that peptide (semipeptoid) is modified, cyclic peptide is modified, N is end modified, C is end modified, peptide bond modification, backbone modifications and residue are modified. Also this specification can deferred to In the technical staff's who instructs the limit of power, test modified antibody or fragment, to estimate Whether their binding characteristic changes.
Equally, change the binding characteristic of antibody, fragment or construct to obtain having more Need the molecule of characteristic, utilizing this paper to provide in technical staff's the limit of power of guidance. Example As, in case identify the antibody with desirable characteristics, then available random or direct mutagenesis produce institute State variant and those variants for the screening desirable characteristics of antibody.
Utilize conventional method known in the art, the technical staff also can be identified for the present invention Additional antibody or its fragment that binding ability is arranged in the composition. For example, additional antibody can be used this The biopanning method that literary composition is described is separated, and wherein is attached to fixing human blood platelets or leukaemia The molecule of cell or cell usually screen concrete phage display library, particularly from leukaemia, The library for preparing in lymthoma or the patients with malignant myeloma.
Utilize conventional method known in the art, the technical staff can also determine to have SEQ ID Resisting of the scFv antibody fragment binding ability of NO:1, SEQ ID NO:2 or SEQ ID NO:3 Body or its fragment. In addition in conjunction with the different molecular or the epi-position that participate in inflammation, for example PSGL-1, Fibrinogen γ ', GP1B, heparin, LUM, complement compound 4 (CC4), The antibody of interalpha inhibitor and factor also can utilize routine side known in the art Method is determined. Utilize conventional method, those skilled in the art can also determine in conjunction with at least a The additional antibody of the epi-position that exists on participation inflammation or the tumour generation cell type, described cell Type comprise B-CLL cell, T-ALL cell, AML cell, B-leukaemia, Multiple myeloma cells and transitional cell. In addition, available conventional method measure in conjunction with lipid, The antibody of sugar, peptide, glycolipid, glycoprotein, lipoprotein and/or lipopolysaccharide molecule epi-position, wherein Described epi-position preferably has at least one sulphation part. Perhaps, also available conventional method is surveyed The antibody of fixed and two or more epi-position cross reactions, each epi-position has one or more sulphur The acidifying tyrosine residue and contain two or more acidic amino acids at least one cluster, its Example is PSGL-1. For example can determine with biosensor analysis in conjunction with data, for example with the merchant The biology sensor of industryization, BIACORE (Piscataway, N.J.) (Myszka, J.Mol. Recognition, 12:279-84 (1999); Malmborg﹠Borrebaeck, J.Immunol. Meth., 183:7-13 (1999)).
The invention provides scFv antibody. ScFv used herein is defined as by can be identical or not The molecule that human antibody heavy chain variable region together and human antibody light chain variable region consist of, and wherein Described variable region of heavy chain connect, connect, merge covalently bound or associate described light chain can Become the district.
The scFv construct can be the scFv molecule polymer (for example dimer, tripolymer, The tetramers etc.), it is in conjunction with one or more antibody hypervariable region. For selective and/or specificity Be attached to the target cell that is conducive to other cell, all scFv source property constructs and fragment are all protected Stay enhancing in conjunction with feature. In conjunction with selective and/or specificity mainly determined by the hypervariable region. Can make up antibody of the present invention to be folded into multivalence Fv type, may improve binding affinity and Specificity, and be increased in half life in the blood.
Other people design and have prepared the scFv of multivalence type. A kind of method is to connect two with joint Individual scFv. Another method comprises utilizes disulfide bond to connect between two scFv. The simplest The method for preparing dimerization or trimerization Fv is by Holliger etc., PNAS 90:6444-48 (1993) With Kortt etc., Protein Eng.10:423-33 (1997) report. Designed a kind of this The method of sample, by one section sequence of interpolation FOS and JUN protein region, between them, At the terminal leucine zipper structure that forms of the C of scFv, the dimer (Kostelny of preparation scFv Deng, J Immunol.148 (5): 1547-53 (1992); De Kruif etc., J Biol Chem. 271 (13): 7630-34 (1996)). Designed another kind of method, by at the C of scFv end Add the Streptavidin coded sequence, the preparation tetramer. Streptavidin is by 4 subunit groups Become, when the scFv-Streptavidin was folding, 4 subunit self-controls formed the tetramer like this (Kipriyanov etc., Hum Antibodies Hybridomas 6 (3): 93-101 (1995)). In another method, in target protein, introduce free cysteine, the preparation dimer, Tripolymer and the tetramer. Have variable number (2-4) maleimide groups based on The cross-over joint of peptide can be used for interconnection target protein and free cysteine (Cochran Deng, Immunity 12 (3): 241-50 (2000)).
In this system, it is many to show to design phage library (described above such as this paper) Plant the scFv of the unit price form that can be folded into antibody Fv district, in addition, also herein above Discuss, described construct is suitable for bacterial expression. The scFv of genetic engineering comprises by connecting Variable region of heavy chain and light chain variables that the flexible peptide spacer region of 15 amino acid of code of sequeling connects The district. Preferred spacer region is (Gly4Ser) 3 The length of this spacer region is together with its amino Acid, component provide non-large volume spacer region together, so that VHDistrict and VLThe district is folded into its target and carries Functional Fv domain for effective combination.
The length that changes described spacer region is that another forms dimer, tripolymer and triamers (usually referring to respectively in the art bifunctional antibody, three function antibodies and four function antibodies) Method for optimizing. Dimer can shorten to common at the spacer region that connects two variable chains of scFv Form under the condition of 5-12 amino acid residue. The spacer region of this shortening can stop identical branch Two variable chains of son are folded into functional Fv domain. And described domain is forced to and another The complementary structure territory pairing of individual molecule produces two binding structural domains. A preferred side In the method, only have 5 amino acid whose spacer region (Gly4Ser) being used for bifunctional antibody makes up. This Dimer can form and keeps by two identical scFv or by two different scFv colonies What parent scFv was selective and/or specificity strengthens is strong in conjunction with combination active and/or that demonstration increases Degree or compatibility.
By similar mode, three function antibodies can be at the spacer region that connects two variable chains of scFv Shorten under the condition that usually is less than 5 amino acid residues and form, it stops same molecular Article two, variable chains is folded into functional Fv domain. And three independent scFv molecular association shapes Become tripolymer. In a preferred method, three function antibodies are by removing this flexibility fully Spacer region obtains. This three function antibody can be by three identical scFv, perhaps by two or three Individual different scFv colony forms, and keeps the selective and/or specificity enhancing of parent scFv In conjunction with active and/or show bond strength or the compatibility that increases.
Four function antibodies can shorten at the spacer region that connects two variable chains of scFv equally usually Be less than under the condition of 5 amino acid residues and form, it stops two variable chains of same molecular Be folded into functional Fv domain. And four independent scFv molecular associations form the tetramer. This four function antibody can be by four identical scFv, perhaps by 1-4 different scFv colony Individual unit forms, and it is alive to keep parent scFv combination selective and/or that specificity strengthens The property and/or the bond strength or the compatibility that show to increase. Usually be less than 5 at described spacer region Under the condition of amino acid residue length, whether three function antibodies or four function antibodies form, and depend on The amino acid sequence of concrete scFv and reaction condition in mixture.
Can in protokaryon or eukaryotic expression system, produce antibody, antibody fragment or its construct, Peptide, polypeptide, protein and fragment thereof and construct. In prokaryotic system or eukaryotic system, produce The method of giving birth to antibody and fragment is well known in the art.
Eukaryotic cell system such as definition among the present invention and discussion, refers to pass through genetic engineering Method produces the expression system of peptide or polypeptide, and wherein host cell is eukaryotic. The eucaryon table The system of reaching can be mammlian system, and the peptide that produces in mammalian expression systems Or polypeptide, behind the purifying, preferably substantially there is not the mammal pollutant. Other utilizes eukaryotic expression The example of system comprises yeast expression system.
The preferred prokaryotic system that produces peptide of the present invention or polypeptide utilizes Escherichia coli (E.coli) to do Host for expression vector. The peptide that in the Escherichia coli system, produces or polypeptide, behind the purifying, Substantially there is not e. coli contamination albumen. Using prokaryotic expression system may cause carrying in the present invention Methionine residues of the terminal increase of the N of some of confession or all sequences. At peptide or polypeptide After generation is expressed described peptide or polypeptide fully, can be by as known in the art, what be fit to Use an example of Aeromonas aminopeptidase under the condition, the methionine of removing the N end is residual Base (United States Patent (USP) the 5th, 763, No. 215).
Can also there be one can insert or connected according to antibody of the present invention and fragment Sign is to help Preparation and identification and diagnosis. This sign can get on from described molecule after a while Remove. The sign example that uses comprises: AU1, AU5, BTag, c-myc, FLAG, Glu-Glu, HA, His6, HSV, HTTPHH, IRS, KT3, C albumen, S-TAG , T7, V5 And VSV-G (Jarvik and Telmer, Ann.Rev.Gen., 32,601-18 (1998)). This mark Will is c-myc or KAK preferably.
Can use any suitable medicine in the present composition. This medicine has anti-usually Cancer activity, antimetastatic activity, anti-leukocythemia liveness, anti-disease activity, activity of anti-adhesion, Anti-thrombosis activity, anti-ISR activity, anti-autoimmunity activity, anti-aggregation activity, Antibacterial activity, antiviral activity or anti-inflammatory activity. Therefore, the medicine of the present composition Can be anticarcinogen, antineoplastic, antiviral agent, antimetastatic agents, anti-inflammatory agent, antithrombotic Form medicine, anti-restenosis, anti-gathering medicine, anti-autoimmunity medicine, antisticking medicine, the anti-heart Angiosis medicine or other disease-resistant medicine or medical substance. Medical substance refers to for prophylactic treatment Or diagnose mammiferous medicine, described mammal include but not limited to people, ox, horse, Pig, mouse, dog, cat or any other warm-blooded animal.
The example of this medicine includes but not limited to antiviral agent, comprises ACV, former times more Luo Wei and Zidovudine; Antithrombotic drug/anti-restenosis comprises Cilostazol, reaches liver Plain sodium, Reviparin Sodium and aspirin; Anti-inflammatory agent, comprise Zaltoprofen, pranoprofen, Drogelor, acetyl salicylic acid 17, Diclofenac, brufen, Dexibuprofen, sulindac, Naproxen, ammonia tolmetin, celecoxib, Indomethacin, rofecoxib, aulin; Anti-autoimmunity medicine, comprise leflunomide, denileukin diftitox, subreum, WinRho SDF, Remove fine glycosides and endoxan; Antisticking medicine/anti-gathering medicine comprises limaprost, chlorine Crow The Meng and hyaluronic acid.
Other illustrative drug comprise cis-platinum, taxol, Calicheamicin, vincristine, Cytarabine (Ara-C), endoxan, metacortandracin, fludarabine, Chlorambucil, dried Disturb plain α, hydroxycarbamide, Temozolomide, Thalidomide and bleomycin and their derivative and Compound. Preferred described medicine is the anthracycline or derivatives thereof, and more preferably described medicine is Doxorubicin (adriamycin), daunorubicin, idarubicin, morpholine Doxorubicin, morpholine soften red Mycin, methoxy morpholine Doxorubicin or derivatives thereof and combination, most preferably described medicine is many Gentle than star (adriamycin).
Anticarcinogen is the medicine with active anticancer. For example, anticarcinogen comprises inhibition or stops The medicine of cancer cell or immature front growth of cancer cells, kill cancer cell or front cancer cell Medicine increases cancer cell or front cancer cell to the medicine of other anti-cancer drugs susceptibility, and suppresses The medicine of cancer metastasis. In the present invention, anticarcinogen also can be to have anti-angiogenic generation Active medicine forms thereby prevent, suppress, postpone or stop tumor vessel. Suppress cancer The growth of cell comprises, for example (i) prevention is carcinous or the metastatic growth, (ii) slows down carcinous or turns to The growth of moving property, (iii) when allowing the cancer cell complete sum live, the growth course of always preventing cancer cell Or transfer process, (iv) contacting of interfere with cancer cells and microenvironment, or (v) kill cancer cell.
Antileukemia is the medicine with anti-leukocythemia liveness. For example, anti-leukocythemia cartridge bag Draw together the medicine that suppresses or stop leukaemia or immature Preleukemia Growth of Cells, kill The medicine of dead leukaemia or Preleukemia cell, increase leukaemia or Preleukemia Cell is to the medicine of the sensitiveness of other anti-leukemia medicine and suppress that the leukaemia shifts Medicine. In the present invention, antileukemia also can be the medicine with anti-angiogenesis activity Thing forms thereby prevent, suppress, postpone or stop tumor vessel. The life of inhibition cancer cell Long comprising, for example (i) prevention is carcinous or the metastatic growth, (ii) slows down carcinous or the metastatic growth, (iii) when allowing the cancer cell complete sum live, always prevent the growth course of cancer cell or shifted Journey, (iv) contacting of interfere with cancer cells and microenvironment, or (v) kill cancer cell.
Can be used for connecting disease-resistant medicine, anticarcinogen and the anti-leukocythemia of antibody of the present invention and fragment The example of medicine comprises toxin, radio isotope and medical substance. The example of toxin comprises white Tree toxin, PE (PE), PE40, PE38, diphtheria toxin, ricin (WA) Or its trim or derivative. Radioisotopic example comprises and can be used for locating and/or controlling Gamma emitter, positron emitter and the X ray emitter treated, and the β that can be used for treating sends out Beam and alpha emitter. Previously described diagnostic radio isotope also can be used for controlling Treat. The limiting examples of anticarcinogen or antileukemia comprises many gentle mycins (adriamycin), suitable Platinum, taxol, Calicheamicin, vincristine, cytarabine (Ara-C), endoxan, Metacortandracin, daunomycin, darubicin, fludarabine, Chlorambucil, interferon-' alpha ', Hydroxycarbamide, Temozolomide, Thalidomide and bleomycin and derivative thereof, and they Combination or trim.
In addition, disease-resistant medicine, anticarcinogen or antileukemia can also be that growth factor receptors is short of money Anti-dose, it suppresses the stimulation of growth factor receptors by the growth factor receptors part, so presses down Tabulation reaches the Growth of Cells of growth factor receptors. The example of some growth factor receptorses is epidermises Growth factor (EGFR), VEGF (VEGFR), platelet-derived growth because of Son (PDGFR), IGF (IGFR), nerve growth factor (NGFR) and becoming Fibroblast growth factor (FGF).
Antibody of the present invention and fragment thereof can also be chosen wantonly and pharmaceutically effectively carrier association, multiple Close or in conjunction with or put together, merge or connect. The carrier example that the present invention uses comprises that the Portugal is poly-Sugar, lipophilic polymer (such as HPMA) and hydrophilic polymer. Perhaps, can use the fat of modification Plastid, such as the liposome with scFv Y1 molecular modification, Doxil for example, this is a kind of commercially available The liposome that contains a large amount of Doxorubicins. Can prepare and thisly contain that one or more are required Medicine and mix to provide a kind of liposome of high drug antibody ratio with antibody of the present invention. Excellent Selecting described medium or carrier is the liposome that Doxorubicin is modified. Perhaps, but also can be preferred Described medium or carrier are hydrophilic polymer polyethylene glycol (PEG) or glucan.
Perhaps, being connected between antibody or its fragment and medicine can be direct connection.Direct connection between two or more adjacent molecules can produce by the chemical bond of element in the molecule or family of elements group.Described chemical bond can be for example ionic bond, covalent bond, hydrophobic bond, hydrophilic bond, electrostatic bond or hydrogen bond.Described key can be for example amine key, carboxylic key, amido link, hydroxyl bond, peptide bond and/or disulfide bond.Described direct connection is the protease resistant key preferably.
Between peptide and medicine or between peptide and carrier, or can be connected by linker compounds between carrier and medicine.As used in description of the present invention and the claim, linker compounds is defined as the chemical compound that connects two or more parts.Described joint can be a straight chain or ramose.The branch joint chemical compound can by two branches, three branches or four branches or more the chemical compound of multiple-limb form.Among the present invention used linker compounds comprise those be selected from have dicarboxylic acids, the linker compounds of maleimide base hydrazides (malemido hydrazides), PDPH, carboxylic hydrazides and little peptide.
According to the present invention, the more specifically example of linker compounds comprises: (a) dicarboxylic acids, as succinic acid, 1,3-propanedicarboxylic acid and adipic acid; (b) maleimide base hydrazides is as N-[maleimide base caproic acid] hydrazides, 4-[N-maleimide ylmethyl] cyclohexyl-1-carboxylic hydrazides and N-[maleimide base undecanoic acid] hydrazides; (c) PDPH joint is as (3-[2-pyridine radicals two sulfur] propionyl hydrazine) puted together with the sulfydryl reactive protein; (d) be selected from the carboxylic hydrazides of 2-5 carbon atom.
By utilizing the direct link coupled connection of little peptide linker also is useful.For example, free sugar and the direct coupling between scFv at the cancer therapy drug doxorubicin can utilize little peptide to finish.The example of little peptide comprises AU1, AU5, BTag, c-myc, FLAG, Glu-Glu, HA, His6, HSV, HTTPHH, IRS, KT3, C albumen, S-TAG , T7, V5, VSV-G and KAK.
Antibody of the present invention and its fragment can with contrast agent (being also referred to as cue mark) for example radiosiotope combine, put together, compound or associate, and these conjugates can be used for diagnosing and the radiography purpose.Test kit with this radiosiotope-antibody (or fragment) conjugate is provided.
Diagnostic radioisotopic example comprises 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.The preferred paramagnetic ion that radiosiotope is roentgenopaque or any is suitable.
Described cue mark molecule also can be a fluorescent tag molecule.The example of fluorescent tag molecule comprises fluorescein, phycoerythrin or rhodamine or their trim or conjugate.
The antibody or the fragment of puting together with cue mark can be used for diagnosis or monitor disease states.This monitoring can be in vivo, external or stripped carrying out.In vivo or under the situation monitor or diagnose that exsomatizes, contrast agent is preferably physiologically acceptable, because it can not hurt the patient unacceptable level.Acceptable injury level can be utilized as the order of severity of disease and the standard of other optional availability by the clinicist and determine.
The invention provides a kind ofly before treatment, during the treatment or the diagnostic kit of treatment back analyzed in vitro therapeutic effect, this test kit comprises a kind of contrast agent or contrast agent that has the peptide of the present invention that is connected with the cue mark molecule.The present invention also provides this contrast agent is used for diagnosis location and cancer radiography, is the method for tumor imaging more precisely, and described method has the following step: cell is contacted with compositions; (b) measure and the bonded radioactivity of described cell; Therefore (c) presents tumor.
The example of suitable contrast agent comprises fluorescent dye, as FITC, PE or the like, and fluorescin, as green fluorescent protein.Other example comprises can produce the Geigers and the enzyme of discernible variation (as change color) with substrate reactions.
In an example, the contrast agent of described test kit is a fluorescent dye, as FITC, and described test kit provides cancer, be to the relevant for example analysis of the therapeutic effect of leukemia, lymphoma or myeloma of cancer of blood more precisely.Facs analysis is used for determining by the percentage ratio of the painted cell of contrast agent with in each stage of disease, for example when diagnosis, during the treatment, between the catabasis and the staining power during the recurrence.
The present invention also provides the arbitrary present composition of patient that needs by having, thereby alleviates the method for disease effector, prevent disease, treatment disease or inhibition disease process.For example, the invention provides suppress to roll before the cell, inflammation-inhibiting, inhibition autoimmune disease, suppress thrombosis, suppress restenosis, suppress to shift, suppress the growth of tumor cell and/or propagation, inhibition leukaemia's growth and/or propagation, the intravital tumor cell number of inhibition tumor patient increases or suppress the method that the intravital leukaemia's number of leukaemic increases.In addition, the invention provides mortality rate, the mortality rate that increases the leukaemia, the sensitivity that increases the damage of diseased cells enantiopathy medicine that increase tumor cell, increase tumor cell to the sensitivity of anticarcinogen damage or increase the method for leukaemia to the sensitivity of anticarcinogen damage.In addition, the invention provides the method that reduces the intravital tumor cell number of tumor patient, reduces the intravital leukaemia's number of leukaemic.At last, the invention provides the method that inhibition cell-cell, cell-matrix, platelet-substrate, platelet-platelet and/or cell-platelet complex forms, assembles or adhere.
The whole bag of tricks of the present invention preferably carries out with the one or both of subclinical amount administered agents and antibody.Therefore, the invention provides the method for therapeutic treatment, described method comprises patient's (i) antibody that needs are arranged or its fragment and (ii) medicine, and wherein said antibody or one of its fragment and/or described medicine or both give with subclinical effective dose.The effective dose of this application will depend on the general state of the order of severity and patient's autoimmune system of disease.Therapeutic regimen also will be because of the state of disease and patient's situation be different, and usually scope from once heavy dose or continuous infusion to repeatedly medication every day (for example every 4-6 hour), or as the treatment doctor indicated with the disease patient.Yet, should be noted that, the invention is not restricted to any concrete dosage.
In addition, it will be recognized by those skilled in the art that the inventive method comprises with single administration or multiple dosing and give described medicine and antibody, it can be like this, before giving described antibody or its fragment, simultaneously or give described medicine afterwards.Can be before giving described antibody or its fragment, simultaneously or give described medicine afterwards, perhaps take over what its order or arrange administration, vice versa.Therefore, described medicine may reside in the different compositionss with antibody.
Antibody of the present invention, construct, conjugate, conjugate and fragment can have the patient who needs by any suitable method afford.Exemplary method 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, preferably prepare preparation, causing the amount that gives patient's desired composition will be from about 0.1mg to the effective dose of about 1000mg.The amount that more preferably gives desired composition will be at about 1mg between about 500mg.Compositions of the present invention is effective in wide dosage range, and depend on various factors for example disease to be treated concrete condition, based on the Pharmaceutical composition of peptide and polypeptide in the mode of administration of the physicochemical property of patient intravital half life, medicine and Pharmaceutical composition, Pharmaceutical composition, to be treated or diagnosis patient's concrete condition etc., and the treatment doctor thinks other important parameters.
Composition for oral liquid can be any suitable form.Example comprises tablet, liquid preparation, Emulsion, suspensoid, syrup, pill, capsule sheet and capsule.The preparation method of Pharmaceutical composition is well known in the art.Referring to for example Remington, The Science andPractice of Pharmacy, Alfonso R.Gennaro (chief editor) Lippincott, Williams﹠amp; Wilkins (publication).
Also can prepare described Pharmaceutical composition so that regularly discharge, continue release, pulse release or release continuously.Described Pharmaceutical composition also can give in device (for example regularly discharging, continue release, pulse release or continuous releasing device).
Topical can be any suitable form with medicinal compositions, as ointment, ointment, lotion, patch, solution, suspensoid, lyophilized preparation and gel.
Comprise antibody of the present invention, construct, conjugate, conjugate and segmental compositions and can comprise conventional pharmaceutically acceptable diluent, excipient, carrier etc.Tablet, pill, capsule sheet, capsule can comprise conventional excipient such as lactose, starch and magnesium stearate.Suppository can comprise excipient such as wax and glycerol.Injection comprises aseptic apyrogeneity medium such as saline, and can comprise buffer agent, stabilizing agent or antiseptic.Also can use conventional casing.
Provide following examples to help to understand the present invention, still do not indicate and should not be considered as by any way to limit the scope of the invention.Though described concrete reagent and reaction condition, can make amendment to it still belongs to category of the present invention.Therefore, provide following example further to illustrate the present invention.All this paper mention or the list of references described all is attached to herein.
Embodiment
Embodiment 1
The present embodiment inspection is the interaction between chemotherapy and Y1 in the mice that has the MOLT-4 tumor.
At first, SCID mice (Jackson) is used 100mg/kg CTX (Cytoxan-Cyclophosphamide for injection, Mead Johnson) pretreatment.The CTX injection is after 5 days, and MOLT-4 (T leukemia) cell passes through the tail vein with 2 * 10 7Individual cell intravenous (i.v.) inoculation.Mice is divided into 5 treatment groups (13 every group) at random, and beginning in 5 days behind cell inoculation, the as shown in the table treatment.Dox course of treatment or afterwards, with the doxorubicin (Dox) and given two weeks of Y1 therapeutic alliance tumor-bearing mice of inferior suitable dosage.According to the reaction of time-to-live monitoring to treatment.As mentioned above, experimental group is as follows:
Table 1
Group institute administered agents (intravenous injection) therapeutic frequency
Not further treatment----of matched group-
2 doses of amycin group 2mg Dox/kg mices, weekly, the inoculation back the 5th certainly
It beginning
6 doses of Y1 group 0.1mg Y1scFv/ mices, inferior on every Wendesdays, the inoculation back the 5th certainly
It beginning
Sequential therapy group 2mg Dox/kg mice, 2 doses, weekly, the inoculation back the 5th certainly
0.1mg Y1scFv/ mice sky begins then, and is 6 doses then, weekly
Therapeutic alliance group 2mg Dox/kg mice+2 doses, weekly, the inoculation back the 5th certainly
0.1mg Y1scFv/ mice sky begins, and is 6 doses then, inferior on every Wendesdays
The result of Fig. 1 shows, with respect to matched group (MTS 39.08 ± 0.8 days), separately with inferior suitable dosage Dox treatment, the life span of tumor-bearing mice had side effect (mean survival time MST is 33.5+1.68 days).Yet earlier with the Dox treatment, the life span with the mice of Y1 sequential therapy prolongs very significantly then.Though Y1 has surprising influence to the survival rate of tumor-bearing mice separately, the clinical suboptimal dose of Dox+Y1 has best effect.It seems that the survival rate that significantly improves be the result of cooperative effect between chemotherapy and Y1 antibody.
Sequence table
＜110〉Sarvent Medicines Corp. (Savient Pharmaceuticals, Inc.)
＜120〉compositions of being used for the treatment of property treatment and method
<130>10793/73
<140>PCT/US03/20604
<141>06/30/2003
<150>10/189,025
<151>07/01/2002
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<170>FastSEQ?for?Windows?Version?3.0
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<211>277
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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?Ser?Phe?Asp?Asp?Tyr?Gly?Met?Ser?Trp?Val?Arg
45 50 55 60
Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val?Ser?Gly?Ile?Asn?Trp?Asn?Gly?Gly?Ser?Thr
65 70 75 80
Gly?Tyr?Ala?Asp?Ser?Val?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ala?Lys?Asn?Ser
85 90 95 100
Leu?Tyr?Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg
105 110 115 120
Met?Arg?Ala?Pro?Val?Ile?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Arg?Gly?Gly?Gly
125 130 135 140
Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Ser?Glu?Leu?Thr?Gln?Asp?Pro?Ala
145 150 155 160
Val?Ser?Val?Ala?Leu?Gly?Gln?Thr?Val?Arg?Ile?Thr?Cys?Gln?Gly?Asp?Ser?Leu?Arg?Ser
165 170 175 180
Tyr?Tyr?Ala?Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Val?Leu?Val?Ile?Tyr?Gly
185 190 195 200
Lys?Asn?Asn?Arg?Pro?Ser?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Gly?Ser?Ser?Ser?Gly?Asn?Thr
205 210 215 220
Ala?Ser?Leu?Thr?Ile?Thr?Gly?Ala?Gln?Ala?Glu?Asp?Glu?Ala?Asp?Tyr?Tyr?Cys?Asn?Ser
225 230 235 240
Arg?Asp?Ser?Ser?Gly?Asn?His?Val?Val?Phe?Gly?Gly?Gy?Thr?Lys?Leu?Thr?Val?Leu?Gly
245 250 255 260
Ala?Ala?Ala?Glu?Gln?Lys?Leu?Ile?Ser?Glu?Glu?Asp?Leu?Asn?Gly?Ala?Ala
265 270 275
<210>2
<211>278
<212>PRT
＜213〉people (Homo sapiens)
<400>2
Met?Lys?Tyr?Leu?Leu?Pro?Thr?Ala?Ala?Ala?Gly?Leu?Leu?Leu?Leu?Ala?Ala?Gln?Pro?Ala
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?Thr?His?Pro?Tyr?Phe?trp?Val
45 50 55 60
Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val?Ser?Gly?Ile?Asn?Trp?Asn?Gly?Gly?Ser
65 70 75 80
Thr?Gly?Tyr?Ala?Asp?Ser?Val?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ala?Lys?Asn
85 90 95 100
Ser?Leu?Tyr?Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala
105 110 115 120
Arg?Met?Arg?Ala?Pro?Val?Ile?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Arg?Gly?Gly
125 130 135 140
Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Ser?Glu?Leu?Thr?Gln?Asp?Pro
145 150 155 160
Ala?Val?Ser?Val?Ala?Leu?Gly?Gln?Thr?Val?Arg?Ile?Thr?Cys?Gln?Gly?Asp?Ser?Leu?Arg
165 170 175 180
Ser?Tyr?Tyr?Ala?Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Val?Leu?Val?Ile?Tyr
185 190 195 200
Gly?Lys?Asn?Asn?Arg?Pro?Ser?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Gly?Ser?Ser?Ser?Gly?Asn
205 210 215 220
Thr?Ala?Ser?Leu?Thr?Ile?Thr?Gly?Ala?Gln?Ala?Glu?Asp?Glu?Ala?Asp?Tyr?Tyr?Cys?Asn
225 230 235 240
Ser?Arg?Asp?Ser?Ser?Gly?Asn?His?Val?Val?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Thr?Val?Leu
245 250 255 260
Gly?Ala?Ala?Ala?Glu?Gln?Lys?Leu?Ile?Ser?Glu?Glu?Asp?Leu?Asn?Gly?Ala?Ala
265 270 275
<210>3
<211>280
<212>PRT
＜213〉people (Homo sapiens)
<400>3
Met?Lys?Tyr?Leu?Leu?Pro?Thr?Ala?Ala?Ala?Gly?Leu?Leu?Leu?Leu?Ala?Ala?Gln?Pro?Ala
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?Glu?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>4
<211>6
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＜213〉people (Homo sapiens)
<400>4
Met?Arg?Ala?Pro?Val?Ile
5
<210>5
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＜213〉people (Homo sapiens)
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Gly?Ile?Asn?Trp?Asn?Gly?Gly?Ser?Thr?Gly?Tyr?Ala?Asp?Ser?Val?Lys
5 10 15
<210>6
<211>5
<212>PRT
＜213〉people (Homo sapiens)
<400>6
Asp?Tyr?Gly?Met?Ser
5
<210>7
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<212>PRT
＜213〉people (Homo sapiens)
<400>7
Leu?Thr?His?Pro?Tyr?Phe
5
<210>8
<211>8
<212>PRT
＜213〉people (Homo sapiens)
<400>8
Leu?Asn?Pro?Lys?Val?Lys?His?Met
5

Claims (63)

1. one kind comprises the segmental compositions of medicine and antibody or its.
2. the compositions of claim 1, wherein said medicine and described antibody or its fragment are compound.
3. the compositions of claim 1, wherein said medicine combines with described antibody or its fragment.
4. the compositions of claim 1, wherein said medicine is puted together with described antibody or its fragment.
5. the compositions of claim 1, wherein said medicine is subclinical amount.
6. the compositions of claim 5, the subclinical quantity not sufficient of wherein said medicine is with the sensitivity of effective change diseased cells enantiopathy medicine.
7. the compositions of claim 5, the subclinical quantity not sufficient of wherein said medicine are rolled before with effective inhibition cell, inflammation, autoimmune disease, thrombosis, restenosis, transfer or tumor cell or leukaemia's growth and/or propagation.
8. the compositions of claim 5, the subclinical quantity not sufficient of wherein said medicine is with the increase of effective inhibition tumor patient interior tumor cell number or be not enough to effectively suppress the increase of leukemia cell number in leukaemic's body.
9. the compositions of claim 5, the subclinical quantity not sufficient of wherein said medicine is to reduce the intravital tumor cell number of tumor patient or to be not enough to reduce the intravital leukaemia's number of leukaemic.
10. the compositions of claim 5, the subclinical quantity not sufficient of wherein said medicine is to increase tumor cell or leukaemia's mortality rate, be not enough to increase the sensitivity of tumor cell, perhaps be not enough to increase the sensitivity of leukaemia the antileukemia damage to the anticarcinogen damage.
11. the compositions of claim 5, the subclinical quantity not sufficient of wherein said medicine forms, assembles or adhesion with effective inhibition cell-cell, cell-matrix, platelet-substrate, platelet-platelet and/or cell-platelet complex.
12. the compositions of claim 1, wherein said antibody or its fragment are subclinical amount.
13. the compositions of claim 1, wherein said antibody or its fragment have the binding ability of the scFv antibody fragment of SEQ IDNO:1, SEQ ID NO:2 or SEQ ID NO:3.
14. the compositions of claim 1, wherein said antibody or its fragment have the binding ability of peptide or polypeptide, and wherein said peptide or polypeptide comprise first hypervariable region with SEQ ID NO:4.
15. the compositions of claim 14, wherein said peptide or polypeptide also comprise second hypervariable region with SEQ IDNO:5 and/or have the 3rd hypervariable region of SEQ ID NO:6, SEQ ID NO:7 or SEQ IDNO:8.
16. the compositions of claim 1, wherein said antibody or its fragment are scFv or Fab fragment.
17. one kind comprises the segmental compositions of medicine and antibody or its, wherein said antibody or its fragment are in conjunction with length about 3 peptide or polypeptide epitopes to about 126 amino acid residues, and wherein said peptide or polypeptide epitope have at least 2 acidic amino acids and at least one sulphation tyrosine residue.
18. one kind comprises the segmental compositions of medicine and antibody or its, wherein said antibody or its fragment are in conjunction with at least two kinds of different molecules, and described molecule is selected from PSGL-1, fibrinogen γ ', GP1b α, heparin, chamber Dan Baijutang, complement chemical compound 4 (CC4), interalpha inhibitor and thrombinogen.
19. one kind comprises the segmental compositions of medicine and antibody or its, wherein said antibody or its fragment are in conjunction with at least two kinds of different molecules and in conjunction with at least a cell type, described molecule is selected from PSGL-1, fibrinogen γ ', GP1b α, heparin, chamber Dan Baijutang, complement chemical compound 4 (CC4), interalpha inhibitor and thrombinogen, and described cell type is selected from B cell leukemia cell, B-CLL cell, AML cell, multiple myeloma cells and transitional cell.
20. one kind comprises the segmental compositions of medicine and antibody or its, wherein said antibody or its fragment and two or more epi-position cross reaction, each epi-position comprise one or more sulphation tyrosine residues and two or more acidic amino acids at least one cluster.
21. the compositions of claim 1, wherein said medicine are selected from anticarcinogen, antimetastatic agents, antileukemia, disease-resistant medicine, antisticking medicine, antithrombotic drug, anti-restenosis, anti-autoimmune medicine, anti-medicine, antimicrobial drug, antiviral agents and the anti-inflammatory agent assembled.
22. the compositions of claim 21, wherein said medicine are to be selected from following antiviral agents: acyclovir, ganciclovir and zidovudine.
23. the compositions of claim 21, wherein said medicine are to be selected from following antithrombotic drug/anti-restenosis: cilostazol, dalteparin sodium, Reviparin Sodium and aspirin.
24. the compositions of claim 21, wherein said medicine are to be selected from following anti-inflammatory agent: zaltoprofen, pranoprofen, drogelor, aspirin 17, diclofenac, ibuprofen, dexibuprofen, sulindac, naproxen, ammonia tolmetin, celecoxib, indomethacin, rofecoxib and nimesulide.
25. the compositions of claim 21, wherein said medicine are to be selected from following anti-autoimmune medicine: leflunomide, denileukin diftitox, subreum, WinRho SDF, defibrotide and cyclophosphamide.
26. the compositions of claim 21, wherein said medicine are to be selected from following antisticking medicine/anti-medicine of assembling: limaprost, cloricromen and hyaluronic acid.
27. the compositions of claim 21, wherein said medicine is selected from toxin, radiosiotope and medical substance.
28. the compositions of claim 27, wherein said toxin are selected from gelonin, Pseudomonas exotoxin (PE), PE40, PE38, Ricin and their trim and derivant.
29. the compositions of claim 27, wherein said radiosiotope are selected from gamma emitter, positron emitter, X ray emitter, beta emitter and alpha emitter.
30. the compositions of claim 27, wherein said 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.
31. the compositions of claim 27, wherein said medicine are selected from cisplatin, paclitaxel, calicheamicin, vincristine, cytosine arabinoside (Ara-C), cyclophosphamide, prednisone, fludarabine, chlorambucil, interferon-ALPHA, hydroxyurea, temozolomide, Thalidomide and bleomycin and their derivant and conjugate.
32. the compositions of claim 27, wherein said medical substance are the anthracycline or derivatives thereofs.
33. the compositions of claim 32, wherein said medical substance are selected from doxorubicin, daunorubicin, idarubicin, morpholine doxorubicin, morpholine daunorubicin, methoxy morpholine doxorubicin and their derivant and conjugate.
34. the compositions of claim 32, wherein said medical substance are the doxorubicin or derivatives thereofs.
35. the compositions of claim 1, wherein said antibody or its fragment and vehicle or carrier coupling or compound or combine, described vehicle or carrier and more than a kind of drug coupling or compound or combine.
36. the compositions of claim 35, wherein said vehicle or carrier are selected from glucosan, lipophilic polymer, hydrophilic polymer, HPMA and liposome.
37. the compositions of claim 36, wherein said vehicle or carrier are the liposomees that doxorubicin is modified.
38. the compositions of claim 36, wherein said vehicle or carrier are Polyethylene Glycol (PEG) or glucosan.
39. a method that suppresses to roll before the cell, described method comprises the compositions that the patient of needs claim 1 is arranged.
40. a method of inhibiting inflammation, described method comprises the compositions that the patient of needs claim 1 is arranged.
41. a method that suppresses autoimmune disease, described method comprises the compositions that the patient of needs claim 1 is arranged.
42. one kind is suppressed thrombotic method, described method comprises the compositions that the patient of needs claim 1 is arranged.
43. a method that suppresses restenosis, described method comprises the compositions that the patient of needs claim 1 is arranged.
44 1 kinds of methods that suppress to shift, described method comprises the compositions that the patient of needs claim 1 is arranged.
45. a method that suppresses growth of tumour cell and/or propagation, described method comprises the compositions that the patient of needs claim 1 is arranged.
46. a method that increases the death of neoplastic cells rate, described method comprises the compositions that the patient of needs claim 1 is arranged.
47. a method that suppresses leukaemia's growth and/or propagation, described method comprises the compositions that the patient of needs claim 1 is arranged.
48. a method that increases leukaemia's mortality rate, described method comprises the Pharmaceutical composition that the patient of needs claim 1 is arranged.
49. a method that increases the sensitivity of diseased cells enantiopathy medicine damage, described method comprises the compositions that the patient of needs claim 1 is arranged.
50. a method that increases tumor cell to the sensitivity of anticarcinogen damage, described method comprises the compositions that the patient of needs claim 1 is arranged.
51. a method that increases the leukaemia to the sensitivity of anticarcinogen damage, described method comprises the compositions that the patient of needs claim 1 is arranged.
52. one kind is suppressed the method that tumor patient interior tumor cell number increases, described method comprises the compositions that the patient of needs claim 1 is arranged.
53. a method that reduces tumor patient interior tumor cell number, described method comprises the compositions that the patient of needs claim 1 is arranged.
54. one kind is suppressed the method that the leukemia cell number increases in leukaemic's body, described method comprises the compositions that the patient of needs claim 1 is arranged.
55. a method that reduces leukemia cell number in leukaemic's body, described method comprises the compositions that the patient of needs claim 1 is arranged.
56. one kind is suppressed the method that cell-cell, cell-matrix, platelet-substrate, platelet-platelet and/or cell-platelet complex forms, described method comprises the compositions that the patient of needs claim 1 is arranged.
57. a method that suppresses cell-cell, cell-matrix, platelet-substrate, platelet-platelet and/or cell-platelet aggregation, described method comprises the compositions that the patient of needs claim 1 is arranged.
58. a method that suppresses cell-cell, cell-matrix, platelet-substrate, platelet-platelet and/or cell-thrombocyte adhesiveness, described method comprises the compositions that the patient of needs claim 1 is arranged.
59. the method alleviating disease effector, prevent disease, treatment disease or suppress disease process, described method comprises the compositions that the patient of needs claim 1 is arranged.
60. the method for a therapeutic treatment, described method comprises the patient that needs are arranged
(i) antibody or its fragment and
(ii) medicine,
Both give in wherein said antibody or its fragment and/or the described medicine one or they with subclinical amount.
61. the method for claim 60, wherein said antibody or its fragment and described medicine independently give.
62. the method for claim 61 wherein gave described antibody or its fragment before giving described medicine.
63. the method for claim 61 wherein gives described antibody or its fragment after giving described medicine.
CNA03820441XA 2002-07-01 2003-06-30 Compositions and methods for therapeutic treatment Pending CN1678348A (en)

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CN106659784A (en) * 2014-07-08 2017-05-10 桑福德伯纳姆医学研究所 PSGL-1 modulators and uses thereof

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WO2004003144A2 (en) 2002-07-01 2004-01-08 Human Genome Sciences, Inc. Antibodies that specifically bind to reg iv
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