CN1997667A

CN1997667A - Humanized FcgammaRIIB-specific antibodies and methods of use thereof

Info

Publication number: CN1997667A
Application number: CN 200580023322
Authority: CN
Inventors: 莱斯利·S·约翰逊; 黄菱
Original assignee: Macrogenics Inc
Current assignee: Macrogenics Inc
Priority date: 2004-05-10
Filing date: 2005-05-10
Publication date: 2007-07-11

Abstract

The present invention relates to humanized FcgammaRIIB antibodies, fragments, and variants thereof that bind human FcgammaRIIB with a greater affinity than said antibody binds FcgammaRIIA. The invention encompasses the use of the humanized antibodies of the invention for the treatment of any disease related to loss of balance of Fc receptor mediated signaling, such as cancer, autoimmune and inflammatory disease. The invention provides methods of enhancing the therapeutic effect of therapeutic antibodies by administering the humanized antibodies of the invention to enhance the effector function of the therapeutic antibodies. The invention also provides methods of enhancing the efficacy of a vaccine composition by administering the humanized antibodies of the invention. The invention encompasses methods for treating an autoimmune disease and methods for elimination of cancer cells that express FcgammaRIIB.

Description

Humanized FcgammaRIIB-specific antibodies and using method thereof

The application requires the right of priority of U.S. Provisional Application 60/582043 (applying date is on June 21st, 2004) and U.S. Provisional Application 60/569882 (applying date is on May 10th, 2004), and these two applications are quoted in full includes the present invention in.

1. technical field

The present invention relates to the fragment and the variant of humanization Fc γ RIIB antibody, this antibody, they and people Fc γ RIIB bonded avidity are greater than described antibody and Fc γ RIIA bonded avidity.The present invention includes and humanized antibody of the present invention is used for the treatment of any and the receptor-mediated signal of Fc conducts unbalance diseases associated, allergic diseases as cancer (being preferably the B cell malignancies, particularly B cell lymphocytic leukemia or non_hodgkin lymphoma), autoimmune disorder, diseases associated with inflammation or Ig-E mediation.The present invention also comprises humanization Fc γ RIIB antibody or its Fab and other cancer treatment method combined utilization.By giving the effector function that humanized antibody of the present invention strengthens therapeutic antibodies, the invention provides the method for the result of treatment that strengthens therapeutic antibodies.By give humanized antibody of the present invention with vaccine composition, the present invention also provides and has strengthened the method that vaccine composition is tired.

2. background of invention

2.1 Fc acceptor and the effect in immunity system thereof

Interaction between antibody-antigenic compound and the immune system cell produces reaction widely, its scope from effector function (for example antibody dependent cellular cytotoxicity, mast cell degranulation and phagolysis) to immunomodulatory signal (for example regulating lymphocytic propagation and antibody-secreting).All these interactions are to combine with specific cells surface receptor on the hematopoietic cell by the Fc district of antibody or immunocomplex to cause.The multifarious reason of the cell response that antibody and immunocomplex cause is the heterology of Fc receptor structure.The Fc acceptor has the ligand binding domain of structurally associated jointly, infers signal conduction in this land mediated cell.

As the member of immunoglobulin gene superfamily protein, the Fc acceptor is the surface glycoprotein that can partly combine with immunoglobulin molecules Fc.Each member of this family discerns one or more immunoglobulin (Ig) hypotypes by the cog region on the Fc receptor alpha chain.The Fc acceptor is defined by its specificity to the immunoglobulin (Ig) hypotype.The Fc acceptor of IgG is called as Fc γ R, and IgE is called Fc ε R, and IgA is called Fc α R.Different helpers has the Fc acceptor at different isotype antibodies, and the isotype of antibody has determined will to participate in specific reaction by which kind of helper, and (summary is seen Ravetch J.V. etc. 1991, Annu.Rev.Immunol.9:457-92; 2001 Microbes and Infection such as Gerber J.S., 3:131-139; Billadeau D.D. etc. 2002, The Journal of Clinical Investigation, 2 (109): 161-168; Ravetch J.V. etc. 2000, Science, 290:84-89; Ravetch J.V. etc., 2001 Annu.Rev.Immunol.19:275-90; Ravetch J.V.1994, Cell, 78 (4): 553-60).Different Fc acceptors, the cell of expressing them and their isotype specificity are summarized in the table 1 and (select from Immunobiology:The Immune System in Health and Disease, 4th ed.1999, Elsevier ScienceLtd/Garland Publishing, New York).

Fc γ acceptor

Each member in this family is complete membrane glycoprotein, has relevant different lengths extracellular domain, single membrane spaning domain and the cytoplasm intracellular domain of C2 combination with the immunoglobulin-related structure territory.3 known Fc γ Rs acceptors are arranged, be called as Fc γ RI (CD64), Fc γ RII (CD32) and Fc γ RIII (CD16).These three kinds of acceptors are encoded by different genes; But the extensive homology between these three family members shows that they may have the common origin by gene replication.Special concern Fc γ RII of the present invention (CD32).

FcγRII(CD32)

Fc γ rii protein is the complete film glycoprotein of 40KDa, because to monomer I g (10 ⁶M ^-1) avidity low and only combine with compound IgG.This receptor is the Fc γ R of wide expression, is present on all hematopoietic cells that comprise monocyte, scavenger cell, B cell, NK cell, neutrophil leucocyte, mastocyte and thrombocyte.Fc γ RII has only two immunoglobulin-like zones on its immunoglobulin (Ig) marriage chain, thereby more much lower than Fc γ RI to the avidity of IgG.3 kinds of people Fc γ RII genes (Fc γ RII-A, Fc γ RII-B, Fc γ RII-C) are arranged, and they all combine with IgG in aggregation or the immunocomplex.

Obvious different two kinds of functional allos reactions that cause receptor-ligandization of Fc γ RII-A (CD32A) and Fc γ RII-B (CD32B) born of the same parents intracellular domain.Essential difference is: the A isotype causes signal conduction in the cell that causes cell activation (as phagolysis and respiratory burst); And the B isotype causes inhibition signal (for example suppressing the B cell activation).

Signal conduction by Fc γ Rs

After the agluconization, activation and inhibition signal all pass through Fc γ R conversion.The textural difference between the isoacceptor isotype has not caused these distinct reverse functions.Two different structure territories of signal conducting region in the endochylema of acceptor (be called as immunoreceptor tyrosine-based activation motif (ITAM) or immunity receptor tyrosine and suppress motif (ITIM)) are the reasons of differential responses.The cell response of Fc γ R mediation takes place in the different cytoplasm enzyme that is focused on these structure peripheries.The Fc γ R mixture that contains ITAM comprises Fc γ RI, Fc γ RIIA, Fc γ RIIIA, and the mixture that contains ITIM has only Fc γ RIIB.

Human neutrophil leucocyte is expressed Fc γ RIIA gene.By immunocomplex or the crosslinked Fc γ RIIA that gathers of specific antibody ITAM is assembled along the kinases relevant with acceptor, this receptor associated kinase is urged the ITAM phosphorylation.The ITAM phosphorylation is as the kinase whose anchored site of Syk, and the kinase whose activation of this Syk causes downstream substrate (PI for example ₃K) activation.Cell activation causes the release of short inflammatory mediator.

Fc γ RIIB gene is expressed on bone-marrow-derived lymphocyte; It is 96% identical that its extracellular domain and Fc γ RIIA have, and combine with the IgG mixture in indistinguishable mode.Determine this inhibition subclass of Fc γ R according to the existence of ITIM in the Fc γ RIIB born of the same parents internal area.Recently, set up this inhibiting molecular basis.When being total to aglucon with reactivity Fc γ R, ITIM among the Fc γ RIIB by phosphorylation and attract inositol polyphosphate 5 '-the SH2 zone of phosphoesterase (SHIP), SHIP hydrolysis phosphoinositide courier (this phosphoinositide courier is that the tyrosine-kinase enzyme activation owing to the Fc γ R mediation that contains ITAM is released), thus Ca in the cell prevented ⁺⁺Interior stream.Therefore, Fc γ RIIB crosslinked blocked the priming reaction of Fc γ R agluconization and suppressed cell response.Therefore B cell activation, B cell proliferation and antibody-secreting have been ended.

The acceptor in table 1 immunoglobulin (Ig) isotype Fc territory

Acceptor	FcγRI (CD64)	FcγRII-A (CD32)	FcγRII-B2 (CD32)	FcγRII-BI (CD32)	FcγRIII (CD16)	FcεRI	FcαRI (CD89)
Acceptor	FcγRI (CD64)	FcγRII-A (CD32)	FcγRII-B2 (CD32)	FcγRII-BI (CD32)	FcγRIII (CD16)	FcεRI	FcαRI (CD89)	In conjunction with	IgG1 10 ⁸M ^-1	IgG1 2×10 ⁶M ^-1	IgG1 2×10 ⁶M ^-1	IgG1 2×10 ⁶M ^-1	IgG1 5×10 ⁵M ^-1	IgG1 10 ¹⁰M ^-1	IgG1，IgA2 10 ⁷M ^-1
Cell category	Scavenger cell neutrophil leucocyte acidophil granules	Scavenger cell neutrophil leucocyte acidophil granules	Scavenger cell neutrophil leucocyte eosinophilic granulocyte	B cell mastocyte	NK cell eosinophilic granulocyte scavenger cell	The mastocyte eosinophilic granulocyte	Scavenger cell neutrophil leucocyte eosinophilic granulocyte	In conjunction with	IgG1 10 ⁸M ^-1	IgG1 2×10 ⁶M ^-1	IgG1 2×10 ⁶M ^-1	IgG1 2×10 ⁶M ^-1	IgG1 5×10 ⁵M ^-1	IgG1 10 ¹⁰M ^-1	IgG1，IgA2 10 ⁷M ^-1

	Cell dendritic cell	Cell dendritic cell thrombocyte Lang Gehansi cell			The neutrophil leucocyte mastocyte	Basophilic granulocyte
	Cell dendritic cell	Cell dendritic cell thrombocyte Lang Gehansi cell			The neutrophil leucocyte mastocyte	Basophilic granulocyte		The effect of agluconization	The activation of picked-up stimulation respiratory burst kills and wounds induces	The picked-up particle release	Picked-up stimulates inhibition	Do not have picked-up and stimulate inhibition	Kill and wound and induce	Granule secretion	Picked-up kills and wounds induces

2.2 relative disease

2.2.1 cancer

Knurl or tumour are the knurl sex organizations that is produced by unusual not controlled cell growth, and it can be benign or virulent.Innocent tumour is usually located at the part.Malignant tumour is referred to as cancer.Term " pernicious " be often referred to this tumour can attack and destroy contiguous housing construction and diffuse to cause at a distance death (summary is referring to Robbins and Angell, 1976, Basic Pathology, the 2nd edition, W.B.Saunders Co., Philadelphia, pp.68-122).Cancer can produce in many positions of body, and shows difference according to its origin.Cancer cells destroys the body part that they originate from, and is diffused into the other parts of body subsequently, begins new growth and causes more serious destruction in the other parts that are diffused into.

Have every year above 1,200,000 American and develop into cancer.American cancer be second largest deadly because of, and if present trend continue development, cancer may become main causes of death before 2010.Lung cancer and prostate cancer are the U.S. male sex's No.1 cancer killers.Lung cancer and mammary cancer are the No.1 cancer killers of American Women's.There is one can be suffered from cancer by diagnosis sometime among two U.S. male sex in its one's remaining years.There is one can be suffered from cancer by diagnosis sometime in three American Women's in its one's remaining years.

Also do not cure method for cancer at present.Present treatment plan does not have effect often or shows severe side effect such as operation, chemotherapy and radiation

2.2.1.1B cell malignancies

The B cell malignancies includes but not limited to B cell lymphoma and leukemia, and it is the knurl disease that remarkable sickness rate is arranged in the U.S..In the U.S. about 55,000 new lymphoma cases (1998 annual data) there is every year, 25,000 people's death are arranged every year approximately.This has represented 4% of the cancer morbidity in the American population, 4% of whole cancer related mortalities.The Europe of second edition-U.S.'s lymph tumor classification (1994, REAL classification, revision in 1999) is divided into B clone lymphoma, T clone lymphoma or He Jiejin lymphomas according to its source with lymphoma.B clone lymphoma be in the non_hodgkin lymphoma (NHL) that the U.S. diagnoses out common type (Williams, Hematology, the 6th edition. (volume such as Beutler), McGraw Hill 2001).

Lymphocytic leukemia (CLL) is a kind of neoplastic disease, with blood, marrow and lymphoid tissue medium and small, lymphocytic gathering of ripe sample be feature.Sickness rate at U.S. CLL is per 100,000 people, 2.7 examples.Increasing with the age, particularly in the male sex, this risk is the increase of carrying out property.It accounts for 0.8% of all cancers, and is modal one-tenth human leukemia, account for all leukemic 30%.In nearly all case (＞98%), diseased cells all belongs to bone-marrow-derived lymphocyte system.Non-leukemia variant, promptly the small lymphocyte lymphoma accounts for all lymphadenomatous 5-10%, its have with B-CLL patient in histology, morphology and the amynologic characteristic (Williams, 2001) of ill lymphoglandula undistinguishable.

The generation history of chronic lymphocytic leukemia is divided into several stages.In early days, chronic lymphocytic leukemia is a kind of painless disease, is feature with malignant B little, sophisticated, that the survival time of non-functional prolongs.At last, the replicative phase of Malignant B cell shortens, patient's sx.But chemotherapy agents treatment relief of symptoms, but total survival of patient only has the prolongation of minimum level.In the late period of chronic lymphocytic leukemia, be feature with significant anaemia and/or thrombopenia.At this moment, the median of survival be lower than 2 years (Foon etc., 1990, Annals Int.Medicine, 113:525).Because the multiplication rate of cell is very low, thus chronic lymphocytic leukemia treatment has resistance to chemotherapeutics.

Recently, gene expression research has identified some genes of possibility up-regulated expression in the lymphopoiesis disease.A kind of be considered in B cell chronic lymphocytic leukemia (B-CLL) patient and most of non_hodgkin lymphoma patient in to cross the molecule of expressing be CD32B (Alizadeh etc., 2000, Nature, 403:503-511; Rosenwald etc., 2001, J.Exp.Med.184:1639-1647).Yet, the effect of CD32B and unclear in B-CLL because have a report proof CD32B on the B-CLL cell of low per-cent with low density express (Damle etc., 2002, Blood, 99:4087-4093).CD32B is a B clone surface antigen, and it is crossed in the B glucagonoma and expresses the suitable target spot that becomes therapeutic antibodies that makes it.In addition, CD32B belongs to inhibition acceptor classification, and it is in conjunction with transmitting the negativity signal.Therefore the antibody of anti-CD32B should have the function of eliminating tumour cell, and its mechanism comprises the cytotoxic effect (CDC) of complement dependence, cytotoxic effect (ADCC) and the startup apoptosis signal that antibody relies on.Therefore, the high homology of CD32B and its copy CD32A (activatory Fc γ acceptor) can block further that selectivity is discerned a kind of of this molecule but not the antibody of another kind of form produces.

2.2.1.2 cancer therapy

At present, treatment for cancer comprises that operation, chemotherapy, hormonotherapy and/or the radiotherapy of eradicating tumour cell among the patient is (referring to for example, Stockdale, 1998, " Principles of Cancer PatientManagement ", in Scientific American:Medicine, the 3rd volume, Rubenstein and Federman compile, the 12nd chapter, IV part).Treatment for cancer has also comprised biotherapy and immunotherapy.All these treatments all have significant disadvantage to the patient.Operative treatment for example, because patient's state of health may be unsuitable for the patient, or unacceptable.In addition, operation also can not be removed tumor tissues fully.Have only when tumor tissues to show when more responsive more than healthy tissues, radiotherapy is only effectively, but radiotherapy also can cause severe side effect usually.Hormonotherapy is seldom used as single agents, although and effectively, normally after having removed most of cancer cells, other treatment is used to prevent or postpone the recurrence of cancer.Biotherapy/immunotherapy limited amount and may having side effects as fash or swelling, influenza-like symptom, comprises heating, shiver with cold and tired, digestive tube problem or anaphylaxis.

For chemotherapy, there are various chemotherapeutics to can be used for cancer therapy.Most of cancer chemotherapy is to come blocking dna to duplicate and the cytodifferentiation followed works (referring to for example by suppressing DNA biosynthesizing synthetic, that directly or indirectly suppress thymus nucleic acid triphosphoric acid precursor, Gilman etc., Goodman and Gilman ' s:The Pharmacological Basis of Therapeutics, the 8th edition (Pergamom Press, New York, 1990)).These reagent, comprise alkylating reagent, resisting-metabolite and other reagent such as methotrexate and hydroxyurea such as nitrosourea, for example etoposides, camptothecine, bleomycin, Zorubicin, daunorubicin or the like, although must cell cycle not have specificity, but because of it acts on dna replication dna, thereby in S phase killer cell.Other reagent, particularly colchicine and vinca alkaloids such as vinealeucoblastine(VLB) and vincristine(VCR), disturb the assembling of microcosmic to cause mitotic division to be stagnated.Chemotherapy regimen generally includes to unite and gives chemotherapeutics and increase result of treatment.

Though there is multiple chemotherapeutics available, but chemotherapy has many shortcomings (referring to for example, Stockdale, 1998, " Principles Of Cancer Patient Management " in Scientific American Medicine, vol.3, Rubenstein and Federman compile, the 12nd chapter, the 10th part).Nearly all chemotherapeutics all is deleterious, and chemotherapy can cause remarkable and common danger, side effect, comprise serious feel sick, bone marrow depression, immunosuppression etc.In addition, even carry out the Combined Preparation of chemotherapeutics, some tumour cells still have resistance or have developed into resistance described chemotherapeutics.In fact, those cells that the concrete chemotherapeutics that uses in the treatment plan had resistance prove that usually other medicines are also had resistance, even mechanism of action are different from those reagent of the mechanism of action of the medicine that uses in concrete treatment plan.This phenomenon is called polytropism medicine and multi-drug resistance.Therefore, because drug resistance, many cancers are difficult to treat to standard chemotherapy regimen.

The B cell malignancies is used the incompatible treatment of single agents chemotherapy, chemotherapy and/or combination radiotherapy group usually.These treatments can reduce sickness rate and/or improve survival, though they have pronounced side effects.The B cell malignancies is complicated to the reaction of multi-form treatment.For example, at the suitable clinical stage of non_hodgkin lymphoma, regional radiotherapy can produce satisfied treatment.Yet some patients are not reaction but, palindromia and prolong in time and treatment is produced the tool aggressive variant of resistance, particularly described disease.There is the patient of half to die from this disease (Devesa etc., 1987, J.Nat ' l Cancer Inst.79:701) approximately.

The exploratory therapy that is used for the treatment of intractable B glucagonoma comprises from body homology and allogenic marrow or in Transplanted cells and gene therapy.Recently, utilize the immunotherapy of the antigenic monoclonal antibody of target B cell-specific to be incorporated in the treatment of B glucagonoma.Thereby the mono-clonal that is used to guide radionuclide, toxin or other therapeutic agent provides and has optionally discharged this reagent restriction to tumor sites the toxicity of healthy tissues is become possibility.

Substituting cancer therapy there is significant demand, need proves the treatment for cancer that is difficult to treat concerning such as the standard cancer treatments of operation, radiotherapy, chemotherapy and hormonotherapy especially.A kind of promising substituting is immunotherapy, and wherein cancer cells is by cancer antigen-specific antibodies specific target.Main effort has been used to make immune response have specificity, and for example, it is possible (referring to Green M.C. etc., 2000, Cancer Treat Rev., 26:269-286 that hybridoma technology becomes the exploitation of tumor-selective antibody; Weiner LM, 1999, Semin Oncol.26 (suppl.14): 43-51), and in the past few years, food and medicine control office has ratified first and has been used for the monoclonal antibody (MAb) of cancer therapy: the Rituxin (anti-CD 20) that is used for non_hodgkin lymphoma, the Campath (anti-CD 52) that is used for B cell chronic lymphocytic leukemia (B-CLL), and Trastuzumab (Herceptin) [anti-(c-erb-2/HER-2)] (the Suzanne A.Eccles that is used for metastatic breast cancer, 2001, Breast Cancer Res., 3:86-90).NHL and B-CLL are two kinds of modal types of B cell tumour.The verified clinical effectiveness of these antibody, but its use is not to be free from side effects.The potential of antibody mediated effect factor function, for example the cytotoxic effect (" ADCC ") of mediate antibody dependence is the obstacle of this treatment.In addition, for Rituxan and Campath, the patient of half does not react it at least, and the part person of responding may produce resistance to treatment subsequently.

Therefore, need be to the cancer selectable therapy of B cell malignancies particularly, especially for the cancer treatment method of standard and the patient that is difficult to treat such as the new immunotherapy of Rituxan.

2.2.2 inflammatory diseases and autoimmune disorder

Inflammation is that the white corpuscle and the chemical substance of body protects body not to be subjected to the process that infects such as foreign matters such as bacterium and viruses.Usually it is characterized by affected regional pain, swelling, generate heat and redden.The chemical substance that is called cytokine and prostaglandin(PG) is controlled this process, and to discharge in blood or the affected tissue with the self-cascade of controlling in order.The release of this chemical substance increases the blood flow of damage or infected zone, and may cause reddening and generating heat.Some chemical substances cause that body fluid this tissue that bleeds causes swelling.This protectiveness process can excite nerve and cause pain.When these changes only took place in the limited time in the relevant range, this was useful to body.

In autoimmune disease and/or inflammatory diseases, immunity system causes Inflammatory response, the not attack of foreign matter at this moment, and the normal protective immunity of body system attacks and injures himself organizing to produce owing to self-mistakenly.The different autoimmune disease of many kinds is arranged, and it influences body by different way.For example, the brain of multiple sclerosis patients is affected, and Crohn disease patient's enteron aisle is affected, and is affected with synovial membrane, bone and the cartilage in the different joints of patient with rheumatoid arthritis.The destruction of autoimmune disease aggravation, a kind of and multiple body tissue type can cause the misgrowth of organ and the variation of organ dysfunction.Autoimmune disease may only influence a kind of organ and types of organization, also can influence multiple organ and tissue.The autoimmune disease organ and the tissue of influence usually comprises red corpuscle, blood vessel, reticular tissue, internal secretion body of gland (for example, Tiroidina or pancreas), muscle, joint and skin.The example of autoimmune disease includes but not limited to struma lymphomatosa, pernicious anemia, Addison's disease, type i diabetes, rheumatoid arthritis, systemic lupus erythematous, dermatomyositis, xerodermosteosis (Siogren ' s syndrome), dermatomyositis, lupus erythematosus, multiple sclerosis, the autoimmune disease of inner ear, myasthenia gravis, conjunctivo-urethro-synovial syndrome (Reiter ' s syndrome), Graves disease (Graves disease), lupoid hepatitis, familial adenomatous polyposis and ulcerative colitis.

Rheumatoid arthritis (RA) and juvenile rheumatoid arthritis belong to the inflammatory arthritis type.Sacroiliitis is to describe the Essential Terms of arthritis.Some sacroiliitis type (but being not whole) is the result of the inflammation of misleading.Except that rheumatoid arthritis, comprise psoriatic arthritis, conjunctivo-urethro-synovial syndrome, ankylosing spondylitis sacroiliitis and urarthritis with the sacroiliitis of other type of inflammation-related.Rheumatoid arthritis is at the simultaneous chronic arthritis type in joint, body both sides (such as hand, wrist and the knee of both sides).This symmetry helps the sacroiliitis of region class rheumatic arthritis and other type.Except that influencing the joint, rheumatoid arthritis influences skin, eye, lung, heart, blood or nerve sometimes.

Rheumatoid arthritis influences about 1% world population and disables most probably.Rheumatoid arthritis in the U.S. annual nearly 2,900,000 is sent out the patient.Women's morbidity is higher 2 to 3 times than the male sex.The typical age of onset of rheumatoid arthritis at 25 years old between 50.71,000 young Americans suffer from juvenile rheumatoid arthritis (age is 18 years old and following), and girl is ill to be 6 times of boy.

Rheumatoid arthritis is an autoimmune disease, and wherein the immunity system of the body synovial membrane that will secrete synovial membrane liquid in the joint is identified as heterogeneous thing undeservedly.Inflammation take place and the joint in and periarticular cartilage and tissue be damaged or destroy.In cases with severe, this inflammation is diffused into other joint tissue and on every side in the cartilage, corrodes at these positions and destroys bone and cartilage and make joint deformity.Body comes for injured tissues with scar tissue, causes that the IA proper space becomes narrow and bone is merged.That rheumatoid arthritis causes is stiff, swelling, fatigue, anaemia, weight loss, fever and common fiber crops pain.Some common symptons of rheumatoid arthritis comprise lasting one hour or play joint stiffness for more time morning; Specific finger or wrist joint swelling; Periarticular soft tissues swelling; The swelling of both sides, joint.Pain can be followed or do not followed to swelling, and carrying out property destruction or kept the several years constant before worsening.

Rheumatoid arthritis can be diagnosed based on the combination of multiple factor, these factors comprise: the concrete zone and the symmetry in pain joint, exist and play joint stiffness morning, lump and tubercle (rheumatic nodules) under the skin, show the X-line detected result of rheumatoid arthritis, and/or be called the positive findings of the blood testing of the rheumatoid factor.Contain the rheumatoid factor antibody in many (but not being whole) patient with rheumatoid arthritis blood.The rheumatoid factor also may appear among the crowd who does not suffer from rheumatoid arthritis.Other disease can cause that also the rheumatoid factor produces in the blood.Here it is why the diagnosis of rheumatoid arthritis need can not only rely on the reason of the existence of the rheumatoid factor in the blood based on several factors in combination.

The canonical process of this disease is a joint symptom lasting but fluctuation, and after about 10 years, 90% patient can show the structure deteriorate of bone and cartilage.The patient that very little per-cent is arranged is the of short duration disease of eliminating fully, has the patient of another very little per-cent that multi-joint odd-shaped serious disease can take place, and has other performance of this disease sometimes.The inflammatory process causes the bone in joint and cartilage to be etched and to destroy.In rheumatoid arthritis, there is the autoimmunization circulation of the antigen presentation, T cytositimulation, cytokine secretion, synovial fluid cell activation and the destruction of joint that continue.This disease all has and seriously influences individual and society, causes tangible pain, function damage and disables, and the health care expense that needs cost millions of dollar wage is in addition lost (referring to for example, NIH network address and NIAID network address).

At present arthritic treatment is mainly concentrated on anti-inflammatory or inhibitive ability of immunity medicine and alleviate the inflammation in joint.One line medicine of any treatment of arthritis all is antiphlogiston usually, for example acetylsalicylic acid, Ibuprofen BP/EP and Cox-2 inhibitor (for example celecoxib and rofecoxib)." two wires medicine " comprises golden preparation, methotrexate and steroid.Although these all are arthritic ripe treatments, seldom having the patient to pass through the independent treatment of these medicines can rehabilitation.Recently, along with to the going deep into of the understanding of Pathogenesis of Rheumatoid Arthritis, unite the soluble receptors that has used methotrexate and cytokine antibodies or reorganization.For example, the recombinant soluble acceptor of tumour necrosis factor (TNF)-α has been used for uniting to come treatment of arthritis with methotrexate.Yet, utilize methotrexate and patient only to have an appointment and 50% show significant clinical improvements such as the anti-TNF-α reagent treatment of recombinant soluble acceptor.Although accepted treatment, many patients still are difficult to treatment.For patient with rheumatoid arthritis, be difficult to the treatment problem and still exist.The progress that existing treatment has side effect occurred frequently or can not ward off disease fully.Therefore, also do not have the ideal methods of treatment, and can not cure.Need the new therapy of wanting more effective treatment rheumatoid arthritis and other autoimmune disease badly

2.2.3 anaphylaxis

According to the fundamental mechanism that causes the allergic symptom performance, immune-mediated allergy (super quick) reaction is divided into four types (I-IV).Type i allergic reaction is a feature with the vaso-active substance that discharges the IgE mediation, as discharging histamine by mastocyte and basophilic granulocyte.Anaphylactogen bonded IgE and its acceptor at mastocyte and basophil cellular surface are crosslinked, cause the release of these materials and allergic symptom subsequently.When be exposed to anaphylactogen the individual second time of suffering from type i allergic reaction, can produce the IgE antibody of high-caliber this allergen specificity, this is to react to each other because memory B cell and T cell have participated in needed three cells of generation IgE.The high-level IgE antibody that is produced can make the crosslinked increase of mastocyte and the lip-deep IgE acceptor of basophilic granulocyte by anaphylactogen bonded IgE, this causes these cell activations and the pathologic medium is discharged, and this medium causes the clinical manifestation of type i allergic reaction.

Discerned and identified two kinds of acceptors that IgE had different avidity.High-affinity receptor (Fc ε RI) is at mastocyte and basophil surface expression.Low-affinity receptor (Fc ε RII/CD23) is expressed on the cell of many types, comprises B cell, T cell, scavenger cell, eosinophilic granulocyte and Lang Gehansi cell.The IgE acceptor of high-affinity is made of 3 kinds of subunits (α, β and γ chain).Some studies show that, have only the α chain to participate in and the combining of IgE, and β and γ chain (for transmembrane protein or suppressor proteins) are used for signal and conduct.In the anaphylactoid scheme that is being designed for treatment or prevention IgE mediation, the identification IgE required structure that combines with Fc ε RI acceptor on mastocyte and the basophil is very important.For example, illustrating of IgE receptor binding site can be used to identify and block IgE in vivo and acceptor carries peptide or the small molecules that cell combines.

At present, adopt the anaphylaxis of pharmacological agent IgE mediation,, be used for alleviating anaphylactoid related symptoms by what offset vaso-active substance that mastocyte and basophil discharged as antihistaminic and corticosteroids.The antihistaminic of high dosage and corticosteroids medicine have toxic side effects (as central nervous system disorders, constipation etc.).Therefore, the method that needs other treatment type i allergic reaction.

A kind of method for the treatment of the type i allergic reaction disease is solubility (free) the IgE reaction in generation and the blood plasma, receptors bind on blocking-up IgE and mastocyte and the basophil, and this antibody do not combine (being the irritated hope property of their right and wrong) with the IgE of bind receptor.FDA has ratified a kind of like this antibody Xolair.

The anaphylactoid a kind of method that is hopeful most to treat the IgE mediation is the active immunization at the epitope of the non-allergenicity of endogenous IgE.People such as Stanworth (U.S. Patent number 5,601,821) disclose a kind of vaccine strategy, relate to use the C ε H4 structural domain that derives from people IgE combine the allos carrier proteins peptide as allergy vaccine.But this peptide has shown can not induce generation and the interactional antibody of natural soluble IgE.In addition, Hellman (U.S. Patent number 5,653,980) has proposed based on the anti-IgE vaccine composition of C ε H2-C ε H3 total length structural domain (about 220 amino acid) with the fusion of foreign vector albumen.But this anti-IgE vaccine composition induces the antibody of generation very likely to cause anaphylaxis, because verified antibody and the IgE acceptor on mastocyte and the basophil at IgE molecule C ε H2 and some part of C ε H3 structural domain is crosslinked, and cause producing anaphylactoid medium (referring to, Stadler etc. for example, 1993, Int.Arch.Allergy and Immunology 102:121-126).Therefore, still need to treat the anaphylaxis of IgE mediation but the method for induced hypersensitivity antibody not.

The special concern of induced hypersensitivity reaction has been produced the method for another treatment type i allergic reaction, this methods of treatment comprises and gives can induce behind the animal body mimic epitopes (Rudolf that generates anti-IgE polyclonal antibody, Deng, 1998, Journal of Immunology 160:3315-3321).People such as Kricek (international publication number WO97/31948) use monoclonal antibody BSWI7 to screen the phage display peptide library, discern and can simulate the peptide mimic epitopes that forms IgE receptors bind conformation.These mimic epitopess may can be used for inducing interact with the natural IgE of free but not with the interactional polyclonal antibody of the IgE of receptors bind, and the combining of blocking-up IgE and its acceptor.People such as Kriek disclose and any part of IgE molecule homologous peptide mimic epitopes not, and therefore it be different from polypeptide disclosed by the invention.

Prove by investigation, still need to strengthen treatment or prevention result of treatment as the existing method of diseases such as cancer, autoimmune disorder, inflammatory diseases or anaphylaxis to prior art.Particularly, need the reinforcing effect subfunction, especially strengthen the cytotoxicity of the therapeutic antibodies in cancer therapy.Still the method (for example, by Antybody therapy or vaccine therapy) that does not have at present treatment or Ammonium Glycyrrhizate disease.

3. summary of the invention

The invention provides humanization Fc γ RIIB antibody, isolated antibody or its fragment, this antibody or fragment and Fc γ RIIB, particularly people Fc γ RIIB, especially combine with natural people Fc γ RIIB specificity, its avidity combines greater than the specificity of described antibody or its fragment and Fc γ RIIA, particularly people Fc γ RIIA, especially natural people Fc γ RIIA." natural Fc γ RIIB and Fc γ RIIA " used herein refers in cell endogenous expression and is arranged in cell surface or recombinant expressed and be positioned at the Fc γ RIIB or the Fc γ RIIA of cell surface at mammalian cell, rather than be expressed in Fc γ RIIB or Fc γ RIIA in the bacterial cell, neither sex change, isolating Fc γ RIIB or Fc γ RIIA.The humanized antibody that the present invention includes and its Fab derive from and Fc γ RIIB, particularly people Fc γ RIIB, especially natural human Fc γ RIIB bonded antibody, and this bonded avidity combining greater than described antibody or its fragment and Fc γ RIIA, particularly people Fc γ RIIA, especially natural people Fc γ RIIA.In the most preferred embodiment, the present invention relates to humanized 2B6 or 3H7 antibody or its fragment, be preferably its Fab.In the another one preferred embodiment, the present invention relates to humanized 1D5,2El, 2H9,2Dl1 or 1F2 antibody and its fragment, be preferably its Fab.

Preferably, humanized antibody of the present invention combines with the cell foreign lands of natural human Fc γ RIIB.The anti-Fc γ of humanization of the present invention RIIB antibody can have the variable region of heavy chain that contains aminoacid sequence CDR1 (SEQ ID NO.1 or SEQ ID NO.29) and/or CDR2 (SEQ ID NO.2 or SEQ ID NO.30) and/or CDR3 (SEQ ID NO.3 or SEQ ID NO.31), and/or contains the variable region of light chain of aminoacid sequence CDR1 (SEQ ID NO.8 or SEQ ID NO.38) and/or CDR2 (SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 or SEQ ID NO.39) and/or CDR3 (SEQ ID NO.12 or SEQ ID NO.40).

In the another one preferred embodiment, humanized antibody of the present invention comprises the variable region of light chain that contains aminoacid sequence SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22 or SEQ ID NO.46, and/or contain the variable region of heavy chain of aminoacid sequence SEQ ID NO.24 or SEQ ID NO.37 and/or their aminoacid sequence variant.

The present invention specifically provides the cell foreign lands immunologic opsonin bonded humanized antibody with natural people Fc γ RIIB, and described antibody comprises (perhaps, being made up of following) VH CDR1 and VL CDR1; VH CDR1 and VL CDR2; VH CDR1 and VL CDR3; VH CDR2 and VL CDR1; VH CDR2 and VLCDR2; VH CDR2 and VL CDR3; VH CDR3 and VH CDR1; VH CDR3 and VL CDR2; VH CDR3 and VL CDR3; VHl CDR1, VH CDR2 and VL CDR1; VH CDR1, VH CDR2 and VL CDR2; VH CDR1, VH CDR2 and VL CDR3; VH CDR2, VH CDR3 and VLCDR1; VH CDR2, VH CDR3 and VL CDR2; VH CDR2, VH CDR2 and VL CDR3; VH CDR1, VL CDR1 and VL CDR2; VH CDR1, VL CDR1 and VL CDR3; VH CDR2, VL CDR1 and VL CDR2; VH CDR2, VL CDR1 and VL CDR3; VH CDR3, VL CDR1 and VL CDR2; VH CDR3, VL CDR1 and VL CDR3; VH CDR1, VH CDR2, VH CDR3 and VL CDR1; VH CDR1, VH CDR2, VH CDR3 and VL CDR2; VH CDR1, VHCDR2, VH CDR3 and VL CDR3; VH CDR1, VH CDR2, VL CD R1 and VL CDR2; VHCDR1, VH CDR2, VL CDR1 and VL CDR3; VH CDR1, VH CDR3, VL CDR1 and VL CDR2; VH CDR1, VH CDR3, VL CDR1 and VL CDR3; VH CDR2, VH CDR3, VL CD R1 and VL CDR2; VH CDR2, VH CDR3, VL CDR1 and VL CDR3; VH CDR2, VH CDR3, VL CDR2 and VL CDR3; VH CDR1, VH CDR2, VH CDR3, VL CDR1 and VL CDR2; VH CDR1, VH CDR2, VL CDR3, VL CDR1 and VL CDR3; VH CDR1, VH CDR3, VL CDR1, VL CDR2 and VL CDR3; VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3; The arbitrary combination of VH CDR perhaps disclosed herein and VL CDR.

In a specific embodiments, the invention provides a kind of humanization 2B6 antibody, wherein VH zone origin comes from the FR fragment of human VH fragment VHl-18 and JH6 and the CDR zone composition of 2B6 VH, contains aminoacid sequence SED ID NO.24.In the another one specific embodiments, the VL zone that humanization 2B6 antibody further comprises, its CDR zone by human VL fragment VK-A26 and JK4 and 2B6VL is formed, and contains aminoacid sequence SEQ ID NO.18, SEQ ID NO.20 or SEQ ID NO.22.

In a specific embodiments, the invention provides a kind of humanization 3H7 antibody, wherein VH zone origin comes from the CDR zone composition of segmental FR fragment of human VH and 3H7 VH, contains aminoacid sequence SED ID NO.37.In the another one specific embodiments, humanization 3H7 antibody further comprises the VL zone of being made up of the CDR zone of segmental FR fragment of human VL and 3H7 VL, contains aminoacid sequence SEQ ID NO.46.

The invention provides the humanized antibody molecule special to Fc γ RIIB, one or more zones of one or more CDR of (receptor's antibody) heavy chain of people's antibody in this antibody and/or variable region of light chain are replaced by the similar portions of one or more CDR of donor monoclonal antibody, described donor monoclonal antibody and Fc γ RIIB specificity bonded avidity greater than with the combining of Fc γ RIIA, for example, by the clone be 2B6 and 3H7 (the ATCC preserving number is respectively PTA-4591 and PTA-4592) generate with Fc γ RIIB bonded monoclonal antibody,, be 1D5 perhaps by the clone, 2El, 2H9, (the ATCC preserving number is respectively PTA-5958 for 2Dl1 and 1F2, PTA-5961, PTA-5962, PTA-5960 and PTA-5959) monoclonal antibody that generates.In the most preferred embodiment, this humanized antibody and donor mouse antibodies specificity are in conjunction with identical epitope.One of ordinary skill in the art will be understood that the antibody CDR that the present invention includes in general sense transplants.Therefore, donor and receptor's antibody can derive from the antibody of allogenic animal or even same classification or subclass.But more generally, donor and receptor's antibody sources is in animal not of the same race.Typically, donor antibody is the non-human antibody, and as the rodents monoclonal antibody, and receptor's antibody is human antibodies.

In some embodiments, at least one CDR from donor antibody is transplanted on the human antibodies.In the other embodiment, each heavy chain and/or light chain at least two, be preferably whole three CDR and be transplanted on the human antibodies.CDR can comprise Kabat CDR, ring texture CDR or their combination.In some embodiments, the included humanization Fc γ RIIB antibody of the present invention comprises at least one heavy chain of having transplanted CDR and at least one and has transplanted the light chain of CDR.

In a preferred embodiment, the CDR zone of humanization Fc γ RIIB specific antibody derives from mouse anti Fc γ RIIB antibody.In some embodiments, humanized antibody described herein comprises change (alteration), this change includes but not limited to: aminoacid deletion, insertion and the modification of receptor's antibody (being the framework region of human heavy chain and/or variable region of light chain, is that maintenance donor monoclonal antibody binding specificity is necessary).In some embodiments, humanized antibody framework region described herein not necessarily must be made up of the accurate aminoacid sequence of the framework region of natural human antibody-like variable region, but can comprise various changes, include but not limited to change aminoacid deletion, insertion and the modification of humanized antibody character, for example, strengthen the binding characteristic of specific region in the humanized antibody, this zone is the same as same target position with mouse Fc γ RIIB specific antibody.In the most preferred embodiment, for fear of a large amount of introducing non-human framework residues and guarantee humanized antibody minimum immunogenicity in human body, to the change minimum of framework region.In some embodiments, the framework residue derives from people VH fragment VH1-18 and JH6 and/or people VL fragment VK-A26 and JK4.In most preferred embodiment of the present invention, framework region is not changed.Donor monoclonal antibody of the present invention be preferably that 2B6 and 3H7 clone system (the ATCC preserving number is respectively PTA-4591 and PTA-4592) generated with Fc γ RIIB bonded monoclonal antibody, the clone that perhaps serves as reasons is the monoclonal antibody that 1D5,2El, 2H9,2Dl1 and 1F2 (the ATCC preserving number is respectively PTA-5958, PTA-5961, PTA-5962, PTA-5960 and PTA-5959) are generated.

Humanized antibody of the present invention comprises the complete antibody molecule with total length heavy chain and light chain, or its any fragment, for example Fab or (Fab ') ₂The dimer of fragment, heavy chain and light chain, or theirs have specific any minimal segment to Fc γ RIIB, as Fv, SCA (single-chain antibody) or the like.

The present invention includes preparation antibody of the present invention or its segmental method, particularly prepare the method for humanization Fc γ RIIB specific antibody, such Fc γ RIIB specific antibody has Fc γ RIIB enhanced avidity with respect to Fc γ RIIA.The present invention includes any method that is used to prepare polypeptide known in the state of the art, for example, external synthetic, recombinant DNA preparation etc.Preferably, this humanized antibody prepares with recombinant DNA technology.Humanization Fc γ RIIB specific antibody of the present invention can prepare by the recombination immunoglobulin expression technology.The standard method for preparing recombinant humanized antibody of the present invention can comprise: a) by conventional molecular biology method construction of expression vector, this expression vector comprises the operon of encoding antibody heavy chain, wherein keep required CDR of donor antibody binding specificity and least part variable region framework from the Fc γ RIIB monoclonal antibody specific of inhuman immunoglobulin (Ig) such as mouse (for example by the clone be 2B6 and 3H7 (A TCC preserving number is respectively PTA-4591 and PTA-4592) generate with Fc γ RIIB bonded monoclonal antibody, be 1D5 perhaps by the clone, 2El, 2H9, (the ATCC preserving number is respectively PTA-5958 for 2D11 and 1F2, PTA-5961, PTA-5962, PTA-5960 and PTA-5959) monoclonal antibody that generated), and the rest part of antibody is from human normal immunoglobulin, thereby generates the carrier that is used to express the humanization heavy chain of antibody; B) molecular biology method by routine makes up the expression vector of the operon that comprises the encoding antibody light chain, wherein keep required CDR of donor antibody binding specificity and least part variable region framework from the Fc γ RIIB monoclonal antibody of inhuman immunoglobulin (Ig) such as mouse (for example by the clone be 2B6 and 3H7 (the ATCC preserving number is respectively PTA-4591 and PTA-4592) generate with Fc γ RIIB bonded monoclonal antibody, be 1D5 perhaps by the clone, 2El, 2H9, (the ATCC preserving number is respectively PTA-5958 for 2Dl1 and 1F2, PTA-5961, PTA-5962, PTA-5960 and PTA-5959) monoclonal antibody that generated, and the rest part of antibody is from human normal immunoglobulin, thereby generates the carrier that is used to express the humanization light chain of antibody; C) change expression vector over to host cell that host cell generates transfection by conventional molecular biology method, be used to express the anti-Fc γ of humanization RIIB antibody; And d) cultivates transfectional cell by conventional cell culture technology, so that the anti-Fc γ of preparation humanization RIIB antibody.Host cell can be by two expression vector cotransfections of the present invention, and first carrier contains the derive operon of polypeptide of encoding heavy chain, and second carrier contains the operon of coding derived light chain polypeptide.Two carriers can contain different selected markers, but except heavy chain and light chain encoding sequence, the other parts of two carriers are preferably identical.This process provides the equal expression of heavy chain and light chain polypeptide.Alternative, can use the single carrier of encoding heavy chain and light chain polypeptide simultaneously.The encoding sequence of heavy chain and light chain can comprise cDNA or genomic dna or both to be had.The host cell that is used for expressing recombinant antibodies of the present invention can be a bacterial cell, as intestinal bacteria, perhaps, is preferably eukaryotic cell.Preferably, can use mammalian cell, as Chinese hamster ovary cell or HEK-293.The selection of host cell is depended in the selection of expression vector, so that have the expression and the control characteristic of expectation in the host cell of selecting.Being used to make up carrier of the present invention, transfectional cell, to prepare the general method that host cell of the present invention, culturing cell prepare antibody of the present invention etc. all be conventional molecular biology method.Equally, recombinant antibodies of the present invention can carry out purifying by the standard method of prior art once generation, comprises cross flow filter, ammonium sulfate precipitation, affinity column chromatography, gel electrophoresis etc.

In some embodiments, the present invention can adopt cell fusion method to prepare monoclonal antibody, and as U.S. Patent number 5,916,771 disclosed contents, this patent are quoted in full includes the present invention in.In brief, according to this method, the DNA of the required heavy chain (or heavy chain fragment) of encoding is imported in first mammalian host cell, and the DNA of the required light chain (or light chain segments) of encoding is imported in second mammalian host cell.Then, formed the 3rd cell by first host cell that transformed and second host cell that transformed through cytogamy.Before merging first cell and second cell, select by cell transformed according to special desired characteristics (as high level expression).After the cytogamy, the hybrid cell of generation contains and expresses the DNA of the required heavy chain of coding and the DNA of the required light chain of coding, realizes the preparation of many subunit antibodies.

The present invention includes humanized antibody of the present invention and other antibody or its fragment (as people or Humanized monoclonal antibodies) are united use or use attached to it.These other antibody can be with antibody of the present invention at the responding property of characteristic mark (epitope) of disease, perhaps these other antibody have the different specificitys through selecting, for example, the molecule or the cell of human immune system are raised ill cell peripheral.Antibody of the present invention (or its part) can be used as the composition of separate administration and uses with such antibody (or its part), perhaps by conventional chemical or molecular biology method two kinds of preparations is connected the back and uses with single composition forms.In addition, can be by humanized antibody being carried out diagnosis and the therapeutic value that mark increases antibody of the present invention, this mark generates detectable signal (external or body in) or has therapeutic property.Some marks, for example the radioactive nuleus thuja acid can generate detectable signal and have therapeutic property.The example of radioisotope labeling comprises ¹²⁵I, ¹³¹I, ¹⁴C.The example of other detectable mark comprises: fluorescence chromophore for example is used for fluorescein, phycobiliprotein or tetraethylrhodamine that fluorescence microscopy is checked; Enzyme, this enzyme produce into fluorescence or coloured product of detecting by fluorescence, specific absorption, visible light or agglutination reaction, and perhaps its generates the high electron density product that shows by electron microscope technique; The high electron density molecule that perhaps is used for direct or indirect electron microscopic imaging, as ferritin, peroxidase or gold.The therapeutic mark comprises the medicine that is used for the treatment of the cancer knurl, for example methotrexate etc.

The inventive method also comprises the polynucleotide of the humanized antibody of the present invention of encoding.In one embodiment, the invention provides the isolating nucleotide sequence of encoding antibody or its segmental heavy chain or light chain, described antibody or its fragment combine with Fc γ RIIB specificity and avidity greater than with the combining of Fc γ RIIA.The invention still further relates to the carrier that contains described Nucleotide.The present invention further provides the carrier of second nucleic acid molecule of first nucleic acid molecule that contains encoding heavy chain and coding light chain, described heavy chain and light chain be with the avidity of Fc γ RIIB specific combination greater than with Fc γ RIIA bonded antibody or its segmental heavy chain and light chain.In a specific embodiments, described carrier is an expression vector.The carrier that contains code book invention antibody or the host cell of polynucleotide have been the present invention further provides.Preferably, (described antibody is generated by the hybridoma cell line of preservation to the present invention includes encoding antibody heavy chain and light chain, the ATCC deposit number is respectively PTA-4591 and PTA-4592, and perhaps the ATCC deposit number is respectively PTA-5958, PTA-5961, PTA-5962, PTA-5960 and PTA-5959) or the polynucleotide of the part (for example CDRs, variable region etc. and humanized variant thereof) of heavy chain of antibody and light chain.

The present invention includes the Fc γ RIIB that uses in the humanization antibodies specific of the present invention ground detection of biological sample (that is, Fc γ RIIB but not Fc γ RIIA).

Excitability and inhibition Fc acceptor, for example Fc γ RIIA and Fc γ RIIB are the keys of function balance between these acceptors and the suitable cell immune response.The present invention includes and use humanization Antybody therapy of the present invention and the unbalance disease relevant of Fc receptor signal conduction with losing regulation and control.Therefore, humanized antibody of the present invention has the immunoreactive purposes of adjusting, for example, and immune response or anaphylaxis that inhibition is relevant with autoimmunization or inflammatory diseases.Humanized antibody of the present invention can also be used to change some effector function to strengthen for example cytotoxicity of therapeutic antibodies mediation.

Humanized antibody of the present invention is used for the prevention and the treatment of cancer, for example, and in one embodiment as independent preparation for treating.In one embodiment of the invention, humanized antibody of the present invention is used for prevention and treatment B cell malignancies, particularly non_hodgkin lymphoma or lymphocytic leukemia.In specific embodiments, patient's tumour tolerates treatment one or more standards or experimental, particularly tolerates the Rituxan treatment.Method of the present invention can treat, controls, prevents or improve and be used for the B cell disease, as B cell lymphocytic leukemia (B-CLL), non_hodgkin lymphoma, diffuse large B cell lymphoma, the follicular lymphoma with diffuse large B cell lymphoma zone, small lymphocyte lymphoma, cover district's cell lymphoma and diffusivity SCC lymphoma.

In the another one embodiment, the invention provides the application of the Fc γ RIIB specific antibody that closes with therapeutic preparation or drug conjugate.The example of the therapeutic preparation that can close with anti-Fc γ RIIB antibody or its Fab yoke includes but not limited to: cytokine, toxin, radioelement and metabolic antagonist.

In one embodiment, the invention provides humanized antibody and treatment B cell malignancies standard or experimental methods of treatment (as chemotherapy, radioimmunoassay therapy or radiotherapy) unite use.Such combination therapy can strengthen curative effect standard or experimental treatment.The example of uniting the useful especially therapeutic preparation that is used to prevent, treat, control the B cell malignancies with Fc γ RIIB specific antibody or its Fab includes but not limited to: Rituxan, interferon alpha and carcinostatic agent.Can include but not limited to the chemotherapy preparation that Fc γ RIIB specific antibody or its Fab are united use: alkylating agent, metabolic antagonist, natural product and hormone.Conjoint therapy of the present invention uses anti-Fc γ RIIB antibody or its Fab than low dosage and/or less frequency of injection can for B cell malignancies patient, and obtains treatment or preventive effect.

In the another one embodiment, the use of humanization Fc γ RIIB antibody or its Fab has prolonged B cell malignancies patient's survival.

In a preferred embodiment, humanized antibody of the present invention is used for the treatment of and/or prevents melanoma.In the another one embodiment, humanized antibody is used to prevent and/or treat cancer, particularly strengthen having the cytotoxicity of the cancer antigen-specific treatment antibody of cytotoxic activity, the phagolysis of, complement-dependent cell toxicant (" CDC ") activity active or therapeutic antibodies with the cell toxicant (" ADCC ") that strengthens the mediation of tumor cytotoxicity and/or antibody dependent cellular.

The invention provides the method that treatment has cancer patients's tumour of cancer antigen property, described method be included as described patient's administering therapeutic significant quantity with Fc γ RIIB specificity bonded avidity greater than combining with Fc γ RIIA bonded first humanized antibody or its fragment with described cancer antigen-specific and having a Cytotoxic second antibody.The present invention also provides the method for the treatment of cancer knurl in the cancer patients's body with cancer antigen property, described method is included as humanized antibody or its fragment of described patient's administering therapeutic significant quantity, this antibody or its fragment and Fc γ RIIB, particularly natural people Fc γ RIIB specificity combination, its bonded avidity is greater than described antibody or its fragment and Fc γ RIIA, particularly natural people Fc γ RIIA bonded avidity, when antibody is monomer, its constant region further to one or more Fc excitability acceptors for example Fc γ RIIIA have enhanced avidity, this method also comprises using with described cancer antigen-specific and is combined with Cytotoxic antibody.In a specific embodiments, described Fc excitability acceptor is Fc γ RIIIA.

In some embodiments, the present invention includes the antibody that contains with the variable Fc of FcRn bonded district, this bonded avidity is enhanced, thereby cause prolong antibody half life, for example the transformation period is greater than 15 days, be preferably more than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months or greater than 5 months.Though do not limited by particular mechanism of action, newly-generated Fc acceptor (FcRn) plays an important role in plasma half-life at adjusting IgG antibody.PH dependency avidity and its relation between plasma half-life in the mouse body of IgG antibody and FcRn have been confirmed.Antibody of the present invention or its fragment transformation period in Mammals (being preferably the mankind) prolongs and to cause described antibody or antibody fragment higher blood plasma titre in Mammals, thereby and has reduced the frequency of administration of described antibody or antibody fragment and/or reduced the concentration of institute's administration of antibodies or antibody fragment.For example, can prepare antibody or its fragment that the transformation period prolongs in the body by interactional aminoacid sequence between those participation Fc structural domains of modification (for example replace, lack or increase) and the FcRn acceptor.For example, the present invention includes and contain the variant Fc regions territory and (have at least one or a plurality of modification, for example modify one or more amino-acid residue 251-256,285-290,308-314,385-389 and 428-436, or in the modification in 250 and 428 sites) antibody, described modification has strengthened the avidity of antibody and FcRn, referring to people such as Hinton, 2004, J.Biol.Chem.279 (8): 6213-6; PCT publication number WO 97/34631; And WO02/060919, these documents are quoted in full includes the present invention in.

In the another one embodiment, the invention provides a kind of method, be used to strengthen the cytotoxicity of patient's internal antibody mediation of accepting cytotoxic antibody treatment, described method is included as described patient and uses the humanized antibody of the present invention of capacity or its fragment to strengthen the cytotoxicity of described cytotoxic antibody.In the another one embodiment, the invention provides a kind of method, be used to strengthen the cytotoxicity of patient's internal antibody mediation of accepting the cytotoxic antibody treatment, described method is included as described patient and uses the humanized antibody of the present invention of capacity or the cytotoxicity that its fragment strengthens described cytotoxic antibody, when being monomer, this antibody or its fragment also have the avidity of enhanced to Fc inhibition acceptor.In the another one embodiment, the invention provides a kind of method, further comprise and use one or more extra cancer therapy.

The present invention includes humanized antibody of the present invention and any therapeutic antibodies that mediates its result of treatment by cell killing are united use, to add the therapeutic activity of powerful antibody.In a specific embodiments, humanized antibody of the present invention is by strengthening the therapeutic activity that antibody-mediated effector function adds powerful antibody.In the another one embodiment of the present invention, humanized antibody of the present invention by strengthen to the target cancer cells engulf or opsonization is strengthened the therapeutic activity of cytotoxic antibody.In another one embodiment of the present invention, humanized antibody of the present invention adds the therapeutic activity of powerful antibody by the cytotoxicity (" ADCC ") that strengthens the antibody dependent cellular mediation that destroys in the target cancer cells process.In certain embodiments, antibody of the present invention and Fc fusion rotein are united to make and are used for strengthening ADCC.

Though be not subjected to the restriction of particular mechanism of action, the use of uniting of humanized antibody of the present invention and therapeutic antibodies has the enhanced result of treatment, and part is owing to the cell toxicant ability of Fc γ RIIB Humanized antibody specific---remove the scavenger cell of expression inhibiting Fc γ RIIB acceptor.Therefore, can remain the cell of the expression excitability FcgR acceptor of greater concn after every dosage treatment antibody is used.

In some embodiments, the present invention comprises humanized antibody united with the therapeutic antibodies that does not mediate its therapeutic action by cell killing and makes the therapeutic activity that is used for strengthening antibody.In a specific embodiments, the present invention includes humanized antibody of the present invention and the therapeutic apoptosis induction antibody (as anti-Fas antibody) with excitability are united use.Therapeutic apoptosis induction antibody can have specificity to the death receptor (for example, TNFR receptor family member or TRAIL family member) that any apoptosis well known in the prior art is regulated approach.

The present invention includes and use humanized antibody of the present invention to block macrophage-mediated tumor cell invasion and transfer.Humanized antibody of the present invention is used in particular for treating the solid tumor that scavenger cell is invaded profit.By reducing or eliminating the scavenger cell quantity that is positioned at tumor site, antagonism humanized antibody of the present invention is used in particular for control (for example reducing or elimination) tumour cell and shifts.The present invention further comprises effective removing or the immune effector cell of elimination except that the scavenger cell of expressing Fc γ RIIB, for example dendritic cells.Use antibody of the present invention can make effector cell's number reduce by 50%, 60%, 70%, 80%, be preferably 90%, most preferably be 99% to the effective removing or the elimination of immune effector cell.

In some embodiments, the present invention includes the therapeutic antibodies that uses humanized antibody of the present invention to combine tumour antigen with immunologic opsonin and unite use, described tumour antigen is not expressed at tumour cell itself, but expresses in the reactive around and supportive non-malignant cell of tumour, tumor stroma.In a preferred embodiment, humanized antibody of the present invention and immunologic opsonin are combined into the antibody combined use of the tumour antigen (fibroblast activation protein (FAP)) on fibrocyte surface.

The invention provides a kind of method of the patient's of treatment autoimmune disorder, described method comprises one or more humanized antibodies of the present invention to described patient's administering therapeutic significant quantity.The present invention also provides a kind of method of the patient's of treatment autoimmune disorder, and described method further comprises one or more anti-inflammatory preparations and/or one or more immunomodulators to described patient's administering therapeutic significant quantity.

The present invention also provides a kind of method of the patient's of treatment inflammatory diseases, and described method comprises one or more humanized antibodies of the present invention to described patient's administering therapeutic significant quantity.The present invention also provides a kind of method of the patient's of treatment inflammatory diseases, and described method further is included as one or more anti-inflammatory preparations and/or one or more immunomodulators of described patient's administering therapeutic significant quantity.

The invention provides a kind of enhancing patient to the immunoreactive method of vaccine composition, described method comprise to described patient use with Fc γ RIIB specificity bonded avidity greater than with Fc γ RIIA bonded humanized antibody or its Fab, and vaccine composition, described antibody or its fragment are subjected to significant quantity and strengthen the immune response of described patient to described vaccine composition.Humanized antibody of the present invention can be used for strengthening body fluid and/or cell-mediated replying at vaccine composition is antigenic.Antibody of the present invention can be united use with any vaccine well known in the prior art.The present invention includes and use humanized antibody of the present invention to prevent or treat specific disease, in this disease, strengthen and to treat or to prevent this disease or illness effectively specific one or more antigenic immunne response.

The present invention also provides a kind of method that infectious agent is carried out immunotherapy, and this method comprises to the patient who is infected by pathogenic agent (as HIV, HCV or HSV) uses humanized antibody of the present invention, to strengthen conditioning and the phagolysis to infected cell.In the another one embodiment, the present invention includes and use humanized antibody of the present invention to treat septicemia or septic shock.The effect of Fc γ RIIB in septicemia revealed (people such as Clatworthy, 2004, J Exp Med 199:717-723).

The signal that the invention provides the mediation of treatment apoptosis conducts the method for impaired disease (for example cancer, autoimmune disorder).In a specific embodiments, the present invention includes the method for the defective disease of apoptosis of treatment Fas mediation, described method comprises co-administered humanized antibody of the present invention and anti-Fas antibody.

The present invention further provides the method for the anaphylactic disease of IgE mediation in treatment or the prevention patient body, this method is included as the humanized antibody of the present invention of described patient's administering therapeutic significant quantity.The present invention also provides the method for the anaphylactic disease of IgE mediation in a kind of treatment or the prevention patient body, and the humanized antibody of the present invention that this method is included as the co-administered treatment significant quantity of described patient is used for treating or prevent the therapeutic antibodies or the vaccine composition of the anaphylactic disease that IgE mediates with other.

In the another one embodiment, the invention provides a kind of method of the patient's of diagnosis autoimmune disorder, this method comprises: (i) will contact with the humanized antibody of the present invention of significant quantity from described patient's biological specimen; (ii) detect described humanized antibody or its segmental combination, exceed background or standard level, illustrate that described patient has autoimmune disorder if detect the described mark that detects.

The present invention further provides a kind of pharmaceutical composition, this pharmaceutical composition comprises: (i) humanized antibody or its fragment of treatment significant quantity, and it and Fc γ RIIB specificity bonded avidity are greater than it and Fc γ RIIA bonded avidity; (ii) pharmaceutically acceptable carrier.The present invention also provides a kind of pharmaceutical composition, and this pharmaceutical composition comprises: (i) a kind of humanized antibody or its fragment for the treatment of significant quantity, and it and Fc γ RIIB specificity bonded avidity are greater than it and Fc γ RIIA bonded avidity; (ii) with cancer antigen-specific bonded cytotoxic antibody; (iii) pharmaceutically acceptable carrier.

In one embodiment of the invention, the pharmaceutical composition that uses according to the inventive method is provided, described pharmaceutical composition comprises: humanization Fc γ RIIB antibody or its Fab of significant quantity, to prevent, to treat, to control or to improve B cell malignancies or its one or more symptoms; With pharmaceutically acceptable carrier.The present invention also provides the pharmaceutical composition that uses according to the inventive method, and described pharmaceutical composition comprises: humanization Fc γ RIIB antibody or its Fab, the preventative or therapeutic agent and the pharmaceutically acceptable carrier of non-Fc γ RIIB antagonist.

3.1 definition

Term used herein " specificity is in conjunction with Fc γ RIIB " and similar terms refer to combine with Fc γ RIIB or its fragments specific and not with other Fc acceptor (particularly Fc γ RIIA) bonded antibody or its fragment (or any other Fc γ RIIB binding molecule).In addition, one of ordinary skill in the art are appreciated that with Fc γ RIIB specificity bonded antibody and can combine by the variable region.If antibody is to combine with Fc γ RIIB specificity by the variable region, the person of ordinary skill in the field is appreciated that it is not an accumulative, that is, it is a monomer.For example measured by known other assay method of immunoassay, BIAcore or prior art, can combine with lower avidity with other peptide or polypeptide with Fc γ RIIB specificity bonded antibody.Preferably, with Fc γ RIIB or its fragments specific bonded antibody or antibody fragment not with other antigenic cross-reaction.Can identify by other technology known to for example immunoassay, BIAcore or the one of ordinary skill in the art with Fc γ RIIB specificity bonded antibody or antibody fragment.Use for example Western hybridization of experimental technique, radioimmunoassay and enzyme-linked immunosorbent assay (ELISAs) to measure, when antibody or its fragment and Fc γ RIIB bonded avidity greater than with the bonding force of any other cross-reactive antigen the time, can determine that antibody combines with Fc γ RIIB specificity with its fragment.Paul is seen in discussion about antibodies specific, ed., 1989, Fundamental Immunology Second Edition, Raven Press, New York 332-336 page or leaf.

Term used herein " natural Fc γ RIIB " refers to endogenous expression and is present in the Fc γ RIIB of cell surface.In some specific embodiments, " natural Fc γ RIIB " is included in protein recombinant expressed in the mammalian cell.Preferably, natural Fc γ RIIB is not at bacterial cell such as expression in escherichia coli.Most preferred, natural Fc γ RIIB is not sex change, that is, it exists with its biological activity conformation.

Term used herein " natural Fc γ RIIA " refers to endogenous expression and is present in the Fc γ RIIA of cell surface.In some embodiments, " natural Fc γ RIIA " is included in protein recombinant expressed in the mammalian cell.Preferably, natural Fc γ RIIA is not at bacterial cell such as expression in escherichia coli.Most preferred, natural Fc γ RIIA is not sex change, that is, it exists with its biological activity conformation.

Term used herein " antibody " refers to that Fvs (sdFv), intrabody (introbody), anti-idiotype (anti-Id) antibody that monoclonal antibody, multi-specificity antibody, people's antibody, humanized antibody, synthetic antibody, chimeric antibody, camelization (camelized) antibody, strand Fvs (scFv), single-chain antibody, Fab fragment, F (ab ') fragment, disulphide connect (comprise, for example, anti-Id antibody and at anti--anti-Id antibody of antibody of the present invention) and the epitope binding fragment of above-mentioned any antibody.Particularly, antibody comprises the immunocompetence fragment of immunoglobulin molecules and immunoglobulin molecules,, contains the molecule of antigen-binding site that is.Immunoglobulin molecules can be any kind (for example IgG, IgE, IgM, IgD, IgA and IgY), classification (IgG for example ₁, IgG ₂, IgG ₃, IgG ₄, IgA ₁And IgA ₂) or subclass.

Term used herein " B cell malignancies " refers to any B cell lymphocytic hyperplasia sexual abnormality.The B cell malignancies comprises the tumour of B origin of cell.The B cell malignancies includes but not limited to: lymphoma, lymphocytic leukemia, acute lymphocytoblast leukemia, multiple myeloma, He Jiejinshi and Fei Hejiejinshi disease, diffuse large B cell lymphoma, the follicular lymphoma with diffuse large B cell lymphoma zone, small lymphocyte lymphoma, lymphoma mantle cell and diffusivity SCC lymphoma.

Term as used herein " derivative " thus refer to by replace, disappearance or increase the antibody that amino-acid residue has been changed amino acid contained sequence.Term used herein " derivative " also refers to the adorned antibody by the molecule of covalently bound any kind.Such as but not limited to the antibody of modifying in the following manner, for example by known protectiveness/closure group, protein solubility cracking, be bonded to cell ligand or other albumen etc. and carry out glycosylation, acetylize, Pegylation, phosphorylation, amidation, derivatize.Deutero-antibody can carry out chemically modified by the known technology of one of ordinary skill in the art and prepare, and these technology include but not limited to: the metabolism of specificity chemical cracking, acetylize, formylation, tunicamycin is synthetic etc.In addition, the antibody of deriving has or identical functions similar to original antibody.

" derivative " that is used in combination with Fc γ RIIB refers to be changed with Fc γ RIIB polypeptide immune specificity bonded the antibody or the antibody fragment of (by replacement, disappearance or the increase (that is sudden change) of amino-acid residue) herein.In some embodiments, antibody derivatives or its fragment comprise replacement, disappearance or the increase of amino-acid residue in one or more CDR.Compare with the non-antibody of deriving, antibody derivatives can have identical in fact combination, better combination or worse combination.In specific embodiments, 1,2,3,4 or 5 amino-acid residue of CDR is substituted, lacks or increase (i.e. sudden change)." derivative " that is used in combination with Fc γ RIIB also refers to antibody or the antibody fragment with Fc γ RIIB polypeptide immune specificity bonded modified (that is, covalently bound by the molecule and the antibody of any kind) herein.For example (but being not limited to) for example by known protectiveness/closure group, protein solubility cracking, be bonded to antibody or antibody fragment that cell ligand or other albumen etc. carry out glycosylation, acetylize, Pegylation, phosphorylation, amidation, derivatize.Deutero-antibody or antibody fragment can include but not limited to that synthetic grade of specificity chemical cracking, acetylize, formylation, tunicamycin metabolism modify by technology well known by persons skilled in the art.In addition, deutero-antibody or antibody fragment can also contain one or more non-classical amino acid.In one embodiment, antibody derivatives has or identical functions similar to female antibody.In the another one embodiment, antibody derivatives or antibody fragment are compared the activity with change with unaltered antibody.For example, derive antibody or its fragment can combine more closely with epitope or more can resist proteolyzing.

Term used herein " illness " and " disease " can be exchanged use, refer to individual a kind of state.Particularly, term " autoimmune disorder " exchanges with term " autoimmune disorder " and uses, refer to a kind of state of patient, thereby it is characterized in that cell, tissue and/or organ damage that the patient produces the immune response of himself cell, tissue and/or organ.Term " diseases associated with inflammation " exchanges with term " inflammatory conditions " and uses, and refers to a kind of state of patient, it is characterized in that inflammation, is preferably chronic inflammatory diseases.Inflammation may be followed or do not followed to autoimmune disorder.In addition, inflammation can yes or no be caused by autoimmune disorder.Therefore, some disease can have the feature of autoimmunization and inflammation simultaneously.

Term used herein " cancer " refers to result from true tumor or tumour unusual, uncontrolled cell growth.As used herein, cancer comprises leukemia and lymphoma clearly.Term " cancer " refers to a kind of disease, its cell that relates to is had the ability to transfer to distal site and is shown the phenotype characteristics that are different from those non-cancer cells, as forming colony or form tubulose network or net sample matrix in three-dimensional matrix (as soft fine jade matter) in three-dimensional basement membrane or extracellular matrix.Non-cancer cells does not form colony in soft fine jade matter, form independently ball-like structure in three-dimensional basement membrane or extracellular matrix.Although be by various mechanism, cancer cells obtains its distinctive functional capabilities in evolution.These abilities comprise escape engulf, self-indulged, insensitive, the tissue intrusion/transfer of growth signals, unlimited expansion potential and lasting blood vessel generation for the long signal of antibiosis.Term " cancer cells " meaning is to comprise premalignant and the virulent cancer cells.In some embodiments, cancer refers to still remain on partial innocent tumour.In other embodiments, the cancer malignant tumour that refers to invade and destroy adjacent housing construction and spread to remote location.In other embodiments, cancer is relevant with specificity cancer antigen.

" immunomodulator " used herein and variant thereof include but not limited to immunomodulator, refer to regulate the preparation of host immune system.In certain embodiments, immunomodulator is the inhibitive ability of immunity preparation.In some other embodiment, immunomodulator is the immunostimulating preparation.Immunomodulator includes but not limited to: small molecules, peptide, polypeptide, fusion rotein, antibody, inorganic molecule, simulant and organic molecule.

Term used herein " epitope " refers on the antigen molecule antibody specificity bonded zone with it.

Term used herein " fragment " refers to contain at least 5 continuous amino acid residues of the aminoacid sequence of another polypeptide, at least 10 continuous amino acid residues, at least 15 amino-acid residues, at least 20 continuous amino acid residues, at least 25 continuous amino acid residues, at least 40 amino-acid residues, at least 50 continuous amino acid residues, at least 60 continuous amino acid residues, at least 70 amino-acid residues, at least 80 continuous amino acid residues, at least 90 continuous amino acid residues, at least 100 amino-acid residues, at least 125 continuous amino acid residues, at least 150 continuous amino acid residues, at least 175 amino-acid residues, 200 continuous amino acid residues, the perhaps peptide of the aminoacid sequence of at least 250 continuous amino acid residues or polypeptide.In a specific embodiments, polypeptide fragment keeps at least a function of this polypeptide.Preferably, antibody fragment is the epitope binding fragment.

Term used herein " humanized antibody " refers to contain the immunoglobulin (Ig) of people's framework region and one or more CDR from non-human immunoglobulin (Ig) (being generally mouse or rat).Provide the non-human immunoglobulin (Ig) of CDR to be called as " donor ", provide the human immunoglobulin of framework to be called as " receptor ".Constant region not necessarily, if but exist, the constant region with human immunoglobulin is identical basically for they, that is, at least about 85-90%, preferred about 95% or more identical.Therefore, except possible CDR, whole parts of Humanized immunoglobulin basically should be identical with the corresponding section of natural human immunoglobulin sequence." humanized antibody " is the antibody that contains humanization light chain and humanization heavy chain immunoglobulin.For example, humanized antibody does not comprise typical chimeric antibody, because for example the whole variable region of chimeric antibody is a non-human.We can say that donor antibody " humanization " by " humanization " process, because expect resulting humanized antibody and the identical antigen of donor antibodies that CDR is provided.In most situations, humanized antibody is human immunoglobulin (receptor's antibody), wherein receptor's hypervariable region is replaced by the hypervariable region residue with desired specificity, avidity and capacity from non-human species's (donor antibody), and described non-human species is as mouse, rat, rabbit or inhuman primate.In some cases, the framework region of human immunoglobulin (FR) residue is replaced by corresponding non-human residue.In addition, humanized antibody can be included in the residue that does not have in receptor's antibody or the donor antibody.These modifications are used to further optimize the antibody performance.Usually, humanized antibody comprises and is no less than at least one, is typically two variable domains, wherein all or all basically hypervariable regions are corresponding to the hypervariable region of non-human immunoglobulin (Ig), and all or all basically FR are the FR with human immunoglobulin sequence.Humanized antibody also can randomly comprise the constant region of partial immunity sphaeroprotein (Fc) at least, representational is and the constant region part of Fc γ RIIB polypeptide immune specificity bonded human immunoglobulin that this constant region is changed by replacement, disappearance or the increase (i.e. sudden change) of amino-acid residue.In some embodiments, humanized antibody is a deutero-.The amino-acid residue that such humanized antibody has comprised in one or more non-human CDR replaces, lacks or increases.The humanized antibody derivative can have identical with non-deutero-humanized antibody in fact combination, better combination or worse combination.In specific embodiments, 1,2,3,4 or 5 amino-acid residue of CDR is substituted, removes or increase (i.e. sudden change).About the more detailed content of humanized antibody, referring to: european patent number EP 239400, EP 592106 and EP 519596; International publication number WO 91/09967 and WO 93/17105; U.S. Patent number 5225539,5530101,5565332,5585089,5766886 and 6407213; And Padlan, 1991, Molecular Immunology28 (4/5): 489-498; People such as Studnicka, 1994, Protein Engineering 7 (6): 805-814; People such as Roguska, 1994, PNAS 91:969-973; People such as Tan, 2002, J.Immunol 169:1119-25; People such as Caldas, 2000, Protein Eng.13:353-60; People such as Morea, 2000, Methods 20:267-79; People such as Baca, 1997, J.Biol Chem.272:10678-84; People such as Roguska, 1996, Protein Eng.9:895-904; People such as Couto, 1995, Cancer Res.55 (23 Supp): 5973s-5977s; People such as Couto, 1995, CancerRes.55:1717-22; Sandhu, 1994, Gene 150:409-10; People such as Pedersen, 1994, J.MoI Biol.235:959-73; People such as Jones, 1986, Nature 321:522-525; People such as Reichmann, 1988, Nature332:323-329; And Presta, 1992, Curr.Op.Struct.Biol.2:593-596.

Term used herein " hypervariable region " refers to be responsible in the antibody amino-acid residue of conjugated antigen.Hypervariable region comprises from the amino-acid residue of " complementary determining region " or " CDR " (promptly, residue 31-35 (H1) in residue 24-34 (L1) in the variable region of light chain, 50-56 (L2) and 89-97 (L3) and the variable region of heavy chain, 50-65 (H2) and 9 5-102 (H3); People such as Kabat, Sequences of Proteins of Immunological Interest, the 5th edition .Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or from amino-acid residue (that is residue 26-32 (H1), 53-55 (H2) and the 96-101 (H3) in the residue 26-32 (L1) in the variable region of light chain, 50-52 (L2) and 91-96 (L3) and the variable region of heavy chain, of " hypermutation ring "; Chothia and Lesk, 1987, J.MoI Biol.196:901-917).CDR residue for Eph099B-208.261 and Eph099B-233.152 is listed in the table 1." framework region " or " FR " residue is those variable region residues except that the hypervariable region of definition here.

Term used herein " strand Fv " or " scFv " refer to antibody fragment, comprise the VH and the VL structural domain of antibody, and wherein these structural domains are arranged in a polypeptide chain.Generally, the Fv polypeptide also comprises the peptide linker between VH and the VL, makes scFv can form antigen in conjunction with needed structure.About the summary of scFv, referring to: Pluckthun in The Pharmacology of Monoclonal Antibodies, 113 volumes, Rosenburg and Moore eds.Springer-Verlag, New York, 269-315 page or leaf (1994).In specific embodiments, scFv comprises the scFv and the humanized scFv of dual specific.

Term used herein " nucleic acid " and " nucleotide sequence " comprise the analogue of dna molecular (as cDNA or genomic dna), RNA molecule (as mRNA), DNA and RNA molecular combinations or DNA/RNA hybrid molecule and DNA or RNA molecule.Such analogue for example can use that nucleotide analog generates, and described nucleotide analog includes but not limited to inosine or trityl base (tritylated bases).These analogues can also comprise and contain DNA or the RNA molecule of modifying skeleton that described modification skeleton is that molecule brings beneficial property, for example nuclease resistance or enhanced cross-cell membrane ability.Nucleic acid or nucleotide sequence can be strand, two strands, can contain strand and double-stranded part simultaneously, can also comprise triple chain portions, but be preferably double-stranded DNA.

Term used herein " individuality " and " patient " are exchanged use.As used herein, individuality is preferably Mammals, as non-human primate animal (as milk cow, pig, horse, cat, dog, mouse etc.) and primate (as monkey and people), most preferably is the people.

Term used herein " treatment " refers to lack the relevant disease or elimination, minimizing or the improvement of condition symptoms to regulating with Fc receptor signal pathway, or strengthens the result of treatment of another therapy (as therapeutic antibodies, vaccine therapy or prevention).In some embodiments, treatment refers to eradicate, remove, change or control former, the partial or cancerous tissue that shifts by using one or more therapeutic preparations.In certain embodiments, this term refers to minimize or delay the metastasis of cancer by use one or more therapeutic preparations to the patient.In other embodiments, this term refers to remove the cell that causes disease.

Phrase used herein " side effect " comprises not wanting and bad effect of preventative preparation or therapeutic preparation.Undesirable action is normally undesired, but undesired effect must not be bad.Undesirable action from preventative preparation or therapeutic preparation may be deleterious uncomfortable or dangerous.Chemotherapeutical side effect includes but not limited to gastrointestinal toxicity, such as but not limited to: early stage and the diarrhoea that occurs late period and flatulence, nauseating, vomiting, apocleisis, oligoleukocythemia, anaemia, neutrophil leucocyte minimizing, weakness, abdominal cramps, heating, pain, weight loss, dehydration, expiratory dyspnea, insomnia, dizziness, mucositis, xerostomia, renal failure, constipation, N﹠M effect, damage temporarily or permanently, influenza-like symptom, fluid retention and sterile temporarily or permanently to the liver kidney.Radiotherapeutic side effect includes but not limited to: tired, dry and apocleisis.The side effect of biotherapy/immunotherapy includes but not limited to: injection site eruption or swelling, influenza-like symptom (as heating, shiver and tired), digestive tube problem and anaphylaxis.The side effect of hormonotherapy includes but not limited to: feel sick, fertility Issue, depression, poor appetite, eye problem, headache and weight fluctuations.Recurrent other the undesirable side effect of patient also has a lot and is that prior art is known, referring to: Physicians ' Desk Reference (the 56th edition, 2002), the document is quoted in full includes the present invention in.

" treatment significant quantity " used herein refers to be enough to treat or control Fc γ RIIB relative disease or illness is regulated the relevant disease of disappearance with any with Fc receptor signal pathway, or strengthens the consumption of therapeutic preparation of the result of treatment of another therapy (as therapeutic antibodies, vaccine therapy or prevention etc.).The treatment significant quantity can refer to be enough to delay or minimize the therapeutic preparation amount that disease takes place, and for example, delays or minimizes the metastasis of cancer.The treatment significant quantity can also refer to provide the therapeutic preparation amount of treatment benefit in disease treatment or control.In addition, the treatment significant quantity of therapeutic preparation of the present invention refer in disease treatment or control, to provide the treatment benefit independent therapeutic preparation amount or unite the amount of use with other treatment, for example, be enough to strengthen the curative effect of the therapeutic antibodies of treatment or control disease.When using with Fc γ RIIB antibodies of the present invention, " treatment significant quantity " can comprise the result of treatment improving wholistic therapy, reduce or avoid undesired side effect or strengthen another preparation or with the synergistic amount of another preparation.

" preventative preparation " used herein refers to any reagent that can be used in preventing disease or preventing disease recurrence or diffusion.The prevention significant quantity can refer to be enough to prevent the recurrence or the diffusion of proliferative disease (particularly cancer), perhaps stops the patient include but not limited to be easy to develop into proliferative disease that the amount of the preventative preparation of proliferative disease (for example because hereditary element be easy to develop into cancer or be exposed to carcinogenic patient) takes place.The prevention significant quantity can also refer to provide the amount of the preventative preparation that prevents benefit in disease prevention.In addition, about the prevention significant quantity of the preventative preparation of the present invention refer in disease prevention, to provide the prevention benefit independent preventative preparation amount or unite the amount of use with other preparation.When being used in combination with the amount of Fc γ RIIB antibody of the present invention, this term can comprise promote whole prevention or strengthen preventive effect or with the synergistic amount of other preventative preparation (such as but not limited to therapeutic antibodies).In certain embodiments, term " preventative preparation " refers to excitability Fc γ RIIB specific antibody.In other embodiments, term " preventative preparation " refers to antagonism Fc γ RIIB specific antibody.In some other embodiment, term " preventative preparation " refers to chemotherapy, radiotherapy, hormonotherapy, biotherapy (as immunotherapy) and/or the Fc γ RIIB antibody of the present invention of cancer.In other embodiments, can unite the preventative preparation that gives more than one.

Term used herein " control " refers to the beneficial effect that the patient obtains from using preventative preparation or therapeutic preparation, but the healing of not arriving disease.In certain embodiments, use one or more preventative preparations or next " control " disease of therapeutic preparation, be progress or the deterioration that wards off disease to the patient.

Term used herein " prevents from " to refer to ward off disease and take place and/or the generation of recurrence or one or more symptoms by use preventative preparation or therapeutic preparation to the patient.

More than one preventative preparation and/or therapeutic preparation " united use " and refer to use in term used herein.The order of administration of disease (for example, proliferative cell disease, especially cancer) preventative preparation of patient and/or therapeutic preparation " united use " and do not limited in term.To the patient use first the prevention or therapeutic preparation can shift to an earlier date (for example 1 fen kind, 5 fens kinds, 15 fens kinds, 30 fens kinds, 45 fens kinds, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, before 12 weeks), simultaneously, or subsequently (for example 1 fen kind, 5 fens kinds, 15 fens kinds, 30 fens kinds, 45 fens kinds, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, after 12 weeks) in second prevention or the using of therapeutic preparation, described patient once suffered from, suffer from now or easy ill individuality.Preventative preparation or therapeutic preparation give the patient at certain time intervals in order, provide than the better result of treatment of other administering mode so that reagent of the present invention can interact with other reagent.Can unite to jade any extra preventative preparation or therapeutic preparation with any order and other extra preventative preparation or therapeutic preparation.

4. description of drawings

Figure 1A and 1B:1A. amino acid are contrasted.Figure 1A has shown contrasting of mouse 2B6VH, humanization 2B6VHl-18 and human JH6 aminoacid sequence; 1B. amino acid is contrasted, and the figure illustrates mouse 2B6VL, human 2B6VL1, human 2B6VL-2, human 2B6VL-3 and the mankind Contrasting of aminoacid sequence.

Fig. 2: hu2B6HC/ch2B6LC monoclonal antibody and ch2B6 monoclonal antibody combine with Fc γ RIIB's.ELISA measures and dimeric combination of solubility Fc γ RIIB-Fc.The avidity of hu2B6HC/ch2B6LC monoclonal antibody bind receptor is similar to the ch2B6 monoclonal antibody.

Fig. 3: hu2B6LC/ch2B6HC monoclonal antibody, ch2B6LC/hu2B6HC and ch2B6 monoclonal antibody combine with Fc γ RIIB's.ELISA measures and dimeric combination of solubility Fc γ RIIB-Fc.The hu2B6HC/ch2B6LC monoclonal antibody is similar to the ch2B6 monoclonal antibody with the avidity of ch2B6HC/hu2B6LC monoclonal antibody bind receptor.

Fig. 4: the hu2B6 variant combines with FcVRIIB's.Hu2B6N50Y, Hu2B6N50Y, V51A, Ch2B6 and Hu2B6 measure with dimeric the combination by ELISA of solubility Fc γ RIIb-Fc.All monoclonal antibodies are similar to the avidity of receptors bind.

Fig. 5: the hu2B6 variant combines with FcTRIIA's.Hu2B6N50Y, Hu2B6N50Y, V51A, Ch2B6 and Hu2B6 measure with dimeric the combination by ELISA of solubility Fc γ RIIa-Fc.Humanization 2B6 monoclonal antibody selective binding CD32B.All actual data point overlap each other, and therefore represent with closed square.

5. specific embodiments

5.1 FCYRIIB specific antibody

The present invention includes humanized antibody (being preferably humanized monoclonal antibody) or its Fab, this antibody or fragments specific in conjunction with Fc γ RIIB, be preferably human Fc gamma RIIB, most preferably be natural human Fc gamma RIIB, its avidity is greater than described antibody or its fragment affinity in conjunction with Fc γ RIIA, particularly people Fc γ RIIA, especially natural people Fc γ RIIA.Preferably, humanized antibody of the present invention combines with the cell foreign lands of natural people Fc γ RIIB.In certain embodiments, this humanized antibody or its fragment bigger 2 times with Fc γ RIIB bonded avidity than it and Fc γ RIIA bonded avidity, 4 times, 6 times, 10 times, 20 times, 50 times, 100 times, 1000 times, 10 ⁴Doubly, 10 ⁵Doubly, 10 ⁶Doubly, 10 ⁷Doubly or 10 ⁸Doubly.In a specific embodiments, humanized antibody of the present invention derives from the mouse monoclonal antibody of clone 2B6 or 3H7 (the ATCC preserving number is respectively PTA-4591 and PTA-4592) generation, perhaps derives from the monoclonal antibody that clone 1D5,2El, 2H9,2Dl1 and 1F2 (the ATCC preserving number is respectively PTA-595B, PTA-5961, PTA-5962, PTA-5960 and PTA-5959) are generated.Budapest treaty regulation according to the microbial preservation international endorsement that is used for patented procedure, the hybridoma that generates antibody 2B6 and 3H7 is deposited in American type culture collection (10801University Blvd. on August 13rd, 2002, Manassas, VA.20110-2209), specified preserving number is respectively PTA-4591 (generating the hybridoma of 2B6) and PTA-4592 (generating the hybridoma of 3H7), and these hybridomas are included into the present invention with way of reference.Regulation according to budapest treaty, the hybridoma that generates 1D5,2El, 2H9,2Dl1 and 1F2 is deposited in American type culture collection (10801 UniversityBlvd. on May 7th, 2004, Manassas, VA.201 10-2209), specified preserving number is respectively PTA-5958, PTA-5961, PTA-5962, PTA-5960 and PTA-5959, and is included into the present invention by reference.

In the another one embodiment, the present invention includes and use the known or disclosed by the invention method of prior art to detect, only combine and Fc γ RIIA is not had the humanization Fc γ RIIB antibody of avidity with Fc γ RIIB.

In a specific embodiments, the present invention includes the humanized antibody of the CDR that contains 2B6 or 3H7.Particularly comprise a kind of antibody, the variable region of heavy chain of this antibody has aminoacid sequence SEQ ID NO:24, and variable region of light chain has aminoacid sequence SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22.In a specific embodiments, the present invention includes a kind of humanized antibody, the variable region of heavy chain of this antibody has aminoacid sequence SEQ ID NO:37, and variable region of light chain has aminoacid sequence SEQ ID NO:46.In the another one preferred embodiment, humanized antibody of the present invention does not also combine with Fc reactivity acceptor such as Fc γ IIA, Fc γ IIIB etc.In one embodiment, humanization Fc γ RIIB specific antibody of the present invention is not to derive from people (1986 such as Pulford, Immunology, 57:71-76) the monoclonal antibody of disclosed called after KB61, neither derive from people (1996 such as Weinrich, Hybridoma, 15 (2): the 109-6) monoclonal antibody of disclosed called after MAbII8D2.In a specific embodiments, the conjugated antigen epi-position of Fc γ RIIB specific antibody of the present invention not with monoclonal antibody KB61 or II8D2 in conjunction with identical epitope, and/or not with monoclonal antibody KB61 or II8D2 competition.Preferably, humanization Fc γ RIIB specific antibody of the present invention does not combine with aminoacid sequence SDPNFSI corresponding to Fc γ RIIb2 isotype 135-141 position.

The selection of the constant region of humanized antibody of the present invention is relevant with the expectation function of this antibody, and is particularly relevant with the effector function that may need.In some embodiments, the constant region of humanized antibody of the present invention is human IgA, IgE, IgG or IgM structural domain.In a specific embodiments, particularly when humanized antibody of the present invention is intended to treat and need the antibody mediated effect subfunction, used the constant region of IgG constant region, particularly IgG1 and IgG3 isotype.In other selectable embodiment, when humanized antibody of the present invention is intended to therapeutic purpose but does not need the antibody mediated effect subfunction, use IgG2 and IgG4 isotype.In other embodiments, humanized antibody of the present invention contains one or more amino acid modified in the Fc zone, as disclosed in the following patent: people's such as Stavenhagen U.S. Patent application 2005/0037000 and 2005/0064514; In the U.S. Provisional Patent Application of on January 9th, 2003, on March 19th, 2003 and submission on October 23rd, 2003, application number is respectively 60/439498,60/456041 and 60/514549 respectively; And U.S. Patent application 5624821 and 5648260 and the patent of European patent EP 0 307 434; The mode that all these patent documentations are all quoted in full is included into the present invention.

The preferred humanized antibody of the present invention combines with the cell foreign lands of natural human Fc gamma RIIB.The anti-Fc γ of humanization of the present invention RIIB antibody can have: the variable region of heavy chain that contains aminoacid sequence CDR1 (SEQ ID NO.1 or SEQ ID NO.29) and/or CDR2 (SEQ ID NO.2 or SEQ ID NO.30) and/or CDR3 (SEQ ID NO.3 or SEQ ID NO.31); And/or contain the variable region of light chain of aminoacid sequence CDR1 (SEQ ID NO.8 or SEQ ID NO.38) and/or CDR2 (SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 or SEQ ID NO.39) and/or CDR3 (SEQ ID NO.12 or SEQ ID NO.40).

In a specific embodiments, the invention provides humanized 2B6 antibody, wherein the VH zone is made up of following: from human VH fragment VH1-18 (people such as Matsuda, 1998, J.Exp.Med.188:2151062) and JH6 (people such as Ravetch, 1981, Cell 27 (3 Pt.2): FR fragment 583-91), have the CDR zone of one or more 2B6VH of aminoacid sequence SED ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.In one embodiment, 2B6 VH has aminoacid sequence SEQ ID NO.24.In the another one specific embodiments, humanized 2B6 antibody further comprises: the VL district, by human VL fragment VK-A26 (people such as Lautner-Rieske, 1992, Eur.J.Immunol.22:1023-1029) and JK4 (people such as Hieter, 1982, J.Biol. Chem.257:1516-22) FR fragment is formed; One or more CDR zone of 2B6VL has aminoacid sequence SEQ ID NO:8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12.In one embodiment, 2B6 VL has aminoacid sequence SEQ ID NO.18, SEQ ID NO:20 or SEQ ID NO:22.

In the another one specific embodiments, the invention provides a kind of humanized 3H7 antibody, wherein consisting of of VH zone: from the segmental FR fragment of human VH, and the CDR zone with aminoacid sequence SED ID NO.37 of 3H7VH.In the another one specific embodiments, humanized 3H7 antibody also comprises the VL zone, the consisting of of VL zone: the segmental FR fragment of human VL, and the CDR zone with 3H7VL of aminoacid sequence SEQ ID NO.46.

Particularly, the invention provides cell foreign lands immunologic opsonin bonded humanized antibody with natural human Fc gamma RIIB, described antibody comprises the CDR sequence of (perhaps, being made up of following) 2B6 or 3H7 with following any array mode: VHCDR1 and VL CDR1; VH CDR1 and VL CDR2; VH CDR1 and VL CDR3; VH CDR2 and VL CDR1; VH CDR2 and VL CDR2; VH CDR2 and VLCDR3; VH CDR3 and VH CDR1; VH CDR3 and VL CDR2; VH CDR3 and VL CDR3; VH1 CDR1, VH CDR2 and VL CDR1; VH CDR1, VH CDR2 and VL CDR2; VHCDR1, VH CDR2 and VL CDR3; VH CDR2, VH CDR3 and VL CDR1; VH CDR2, VH CDR3 and VL CDR2; VH CDR2, VH CDR2 and VL CDR3; VH CDR1, VL CDR1 and VL CDR2; VH CDR1, VL CDR1 and VL CDR3; VH CDR2, VL CDR1 and VL CDR2; VH CDR2, VL CDR1 and VL CDR3; VH CDR3, VL CDR1 and VL CDR2; VH CDR3, VL CDR1 and VL CDR3; VH CDR1, VH CDR2, VH CDR3 and VL CDR1; VH CDR1, VH CDR2, VH CDR3 and VL CDR2; VH CDR1, VH CDR2, VH CDR3 and VL CDR3; VH CDR1, VH CDR2, VL CDR1 and VL CDR2; VH CDR1, VH CDR2, VL CDR1 and VL CDR3; VH CDR1, VH CDR3, VL CDR1 and VL CDR2; VH CDR1, VHCDR3, VL CDR1 and VL CDR3; VH CDR2, VH CDR3, VL CDR1 and VL CDR2; VHCDR2, VH CDR3, VL CDR1 and VL CDR3; VH CDR2, VH CDR3, VH CDR2 and VL CDR3; VH CDR1, VH CDR2, VH CDR3, VL CDR1 and VL CDR2; VH CDR1, VH CDR2, VH CDR3, VL CDR1 and VL CDR3; VH CDR1, VH CDR2, VL CDR1, VL CDR2 and VLCDR3; VH CDR1, VH CDR3, VL CDR1, VL CDR2 and VLCDR3; VH CDR2, VH CDR3, VLCDR1, VL CDR2 and VL CDR3; The arbitrary combination of VH CDR perhaps disclosed herein and VL CDR.

The invention provides specific humanized antibody molecule to Fc γ RIIB, wherein, one or more zones of one or more CDR of people's antibody (receptor's antibody) heavy chain and/or variable region of light chain are replaced by the same merit part of one or more CDR of donor monoclonal antibody, described donor monoclonal antibody and FC γ RIIB specificity bonded avidity greater than with the combining of Fc γ RIIA, for example, generate by clone 2B6 and 3H7 (the ATCC preserving number is respectively PTA-4591 and PTA-4592) with Fc γ RIIB bonded monoclonal antibody, perhaps by clone 1D5,2El, 2H9, (the ATCC preserving number is respectively PTA-5958 for 2Dl1 and 1F2, PTA-5961, PTA-5962, PTA-5960 and PTA-5959) monoclonal antibody that generates.In other embodiments, humanized antibody of the present invention and 2B6,3H7,1D5,2El, 2H9,2Dl1 or 1F2 are in conjunction with identical epitope.In the most preferred embodiment, this humanized antibody can be specifically with the donor mouse antibodies in conjunction with identical epitope.One of ordinary skill in the art will be understood that the antibody CDR that the present invention includes ordinary meaning transplants.Therefore, donor and receptor's antibody can derive from the antibody of same species or even same classification or subclass.But more general, donor and receptor's antibody sources is in different plant species.Typically, donor antibody is the non-human antibody, and as the rodents monoclonal antibody, and receptor's antibody is human antibodies.

In some embodiments, the CDR of at least one donor antibody is transplanted on the human antibodies.In other embodiments, at least two of each heavy chain and/or variable region of light chain, be preferably whole three CDR and be transplanted on the human antibodies.CDR can comprise Kabat CDR, ring texture CDR or their combination.In some embodiments, humanization Fc γ RIIB antibody of the present invention contains the heavy chain of at least one CDR transplanting and the light chain that at least one CDR transplants.

In a preferred embodiment, the CDR zone of humanization Fc γ RIIB specific antibody derives from mouse anti Fc γ RIIB antibody.In some embodiments, humanized antibody described herein comprises change, includes but not limited to: aminoacid deletion, insertion and the modification of receptor's antibody (as keeping the framework region of donor necessary human heavy chain of monoclonal antibody binding specificity and/or variable region of light chain).In some embodiments, humanized antibody framework region described herein is not the accurate aminoacid sequence that framework region had that must comprise natural human antibody-like variable region, and can comprise various changes, include but not limited to change aminoacid deletion, insertion and the modification of humanized antibody character, for example, improve the binding characteristic of humanized antibody specific region (special) to the target position identical with mouse Fc γ RIIB specific antibody.In the most preferred embodiment, framework region is made minimum change, purpose is the immunogenicity of avoiding introducing non-human framework residue in a large number and guaranteeing humanized antibody minimum in human body.Donor monoclonal antibody of the present invention is preferably the monoclonal antibody that is generated with Fc γ RIIB bonded clone 2B6 and 3H7 (the ATCC preserving number is respectively PTA-4591 and PTA-4592), the monoclonal antibody that perhaps serve as reasons clone 1D5,2El, 2H9,2Dl1 and 1F2 (the ATCC preserving number is respectively PTA-5958, PTA-5961, PTA-5962, PTA-5960 and PTA-5959) are generated.

In a specific embodiments, the present invention includes the antibody that CDR transplants, itself and Fc γ RIIB specificity bonded avidity combining greater than described antibody and Fc γ RIIA, wherein the variable region of heavy chain of this CDR grafted antibody comprises the framework residue of receptor's antibody and from the residue of donor monoclonal antibody, donor monoclonal antibody and Fc γ RIIB specificity bonded avidity greater than with Fc γ RIIA bonded avidity, for example, clone's monoclonal antibody that 2B6,3H7,1D5,2El, 2H9,2Dl1 or 1F2 generated.In the another one specific embodiments, CDR grafted antibody of the present invention and Fc γ RIIB specificity bonded avidity greater than with the avidity of Fc γ RIIA, this CDR grafted antibody comprises the framework residue that contains receptor's antibody and from the residue of donor monoclonal antibody, this donor monoclonal antibody and Fc γ RIIB specificity bonded avidity greater than with the avidity of Fc γ RIIA, for example, clone's monoclonal antibody that 2B6,3H7,1D5,2El, 2H9,2Dl1 or 1F2 generated.

Humanized antibody of the present invention consist essentially of all at least one, common two variable regions, all or all basically CDR zone be corresponding to the variable region of non-human immunoglobulin (Ig) (being donor antibody) in the variable region, and all or all basically framework regions are the framework regions of human immunoglobulin consensus sequence.Preferably, humanized antibody of the present invention also comprises the constant region of partial immunity sphaeroprotein (Fc) at least, is generally the constant region of human immunoglobulin.The selection of the constant region of humanized antibody of the present invention is relevant with the expectation function of this antibody, and is particularly relevant with the effector function that may need.In some embodiments, the constant region of humanized antibody of the present invention is human IgA, IgE, IgG or IgM structural domain.In a specific embodiments, when humanized antibody of the present invention is intended to treat and need the antibody mediated effect subfunction, used the constant region of IgG constant region, particularly IgG1 and IgG3 isotype.In alternative embodiment, when humanized antibody of the present invention is intended to therapeutic purpose but does not need the antibody mediated effect subfunction, IgG2 and IgG4 isotype have been used.Present invention resides in the Fc zone and contain one or more amino acid modifiedly, this modification changes the effector function of antibody, as people's such as Stavenhagen U.S. Patent Publication 2005/0037000 and 2005/0064514; The United States Patent (USP) provisional application of submitting on January 9th, 2003, on March 19th, 2003 and on October 23rd, 2,003 60/439498,60/456041 and 60/514549 respectively; All these patent documentations are all quoted in full includes the present invention in.

In some embodiments, humanized antibody of the present invention contains the light chain and the variable region of heavy chain at least simultaneously.In other embodiments, humanized antibody of the present invention may further include one or more among CH1 in the heavy chain, hinge area, CH2, CH3 and the CH4.This humanized antibody can be selected from the immunoglobulin (Ig) of any kind of, comprises IgM, IgG, IgD, IgA and IgE, and any isotype, comprises IgG1, IgG2, IgG3 and IgG4.In some embodiments, when the needs humanized antibody had cytotoxic activity, constant region was the constant region of complement-fixing, and the kind of immunoglobulin (Ig) is generally IgG1.In other embodiments, when not needing this cytotoxic activity, constant region can be the constant region of IgG2 class.Humanized antibody of the present invention can comprise the sequence from an above kind or isotype, and the effector function of selecting specific constant region to optimize expectation is the ordinary skill of this area.

The framework of humanized antibody and CDR zone do not need accurately corresponding to parental array, for example the CDR of donor or total framework can by mutagenic treatment, make that the CDR in that site or framework residue are not corresponding with consensus sequence or donor antibody by replacement, insertion or the disappearance of at least one residue.But preferred, such variation is not widely.Usually, at least 75% humanized antibody residue is corresponding to the residue of parent's framework region (FR) and CDR sequence, and more general is 90%, most preferably greater than 95%.Can use the known multiple technologies of prior art to prepare to come humanized antibody, include but not limited to: CDR transplants (european patent number EP 239,400; International publication number WO 91/09967; With U.S. Patent number 5,225,539,5,530,101 and 5,585,089), frosting (veneering) or come to the surface again (resurfacing) (european patent number EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28 (4/5): 489-498; People such as Studnicka, 1994, Protein Engineering 7 (6): 805-814; With people such as Roguska, 1994, Proc.Natl.Acad.Sci.91:969-973), chain replaces disclosed technology (as: U.S. Patent number 6407213,5766886,5585089 in (U.S. Patent number 5,565,332) and other document; International publication number WO 9317105; People such as Tan, 2002, J.Immunol.169:1119-25, people such as Caldas, 2000, Protein Eng.13:353-60; People such as Morea, 2000, Methods 20:267-79; People such as Baca, 1997, J.Biol Chem.272:10678-84; People such as Roguska, 1996, Protein Eng.9:895-904; People such as Couto, 1995, Cancer Res.55 (23 Supp): 5973s-5977s; People such as Couto, 1995, Cancer Res.55:1717-22, Sandhu, 1994, Gene 150:409-10; People such as Pedersen, 1994, J.MoI.Biol.235:959-73; People such as Jones, 1986, Nature 321:522-525; People such as Riechmann, 1988, Nature 332:323; And Presta, 1992, Curr.Op.Struct.Biol.2:593-596).Usually, the framework residue in the framework region will be replaced by the corresponding residue from CDR donor antibody, changes, preferably enhancement antigen combination.These frameworks replace to be discerned by method well known in the prior art, for example, discern antigen in conjunction with important framework residue by the interaction model of making CDR and framework residue, by comparative sequences discern uncommon framework residue on the specific position (referring to: as, people's such as Queen U.S. Patent number 5,585,089; U.S. Patent Publication 2004/0049014 and 2003/0229208; U.S. Patent number 6,350,861; 6,180,370; 5,693,762; 5,693,761; 5,585,089 and 5,530,101, and people such as Riechmann, 1988, Nature332:323; All these documents are all quoted in full includes the present invention in).

In a particular, a kind of activity of humanization of the present invention or the exciting at least Fc γ of its fragment RIIB.In one embodiment of the invention, described activity is to suppress the signal conduction of B-cell receptor mediation.In the another one embodiment, humanization agonistic antibody of the present invention suppresses activation, the increment of B cell, antibody generation, the interior stream of intracellular calcium of B cell, the cell cycle progression of B cell, or suppresses the activity of one or more downstream signaling molecules in the Fc γ RIIB signal transduction path.In the another one embodiment, phosphorylation or SHIP that humanization agonistic antibody of the present invention strengthens Fc γ RIIB raise.In another one embodiment of the present invention, map kinase activity or Akt in the signal transduction path of humanization agonistic antibody inhibition B-cell receptor mediation raise.In the another one embodiment, humanization agonistic antibody of the present invention activates the inhibition to the conduction of Fc ε RI signal of Fc γ RIIB mediation.In a particular, described humanized antibody suppresses the activation of Fc ε RI inductive mastocyte, calcium mobilization, takes off particle, cytokine generation or thrombotonin release.In the another one embodiment, humanization agonistic antibody of the present invention stimulates Fc γ RIIB phosphorylation, stimulate SHIP raise, stimulate the SHIP phosphorylation and with the activation that combines or suppress map kinase family member (for example Erk1, Erk2, JNK, p38 etc.) of Shc.In the another one embodiment, humanization agonistic antibody of the present invention strengthens p62 ^DokTyrosine phosphorylation and with the combining of SHIP and rasGAP.In the another one embodiment, humanization agonistic antibody of the present invention suppresses the monocyte of Fc γ R mediation or the phagolysis of scavenger cell.

In another embodiment, at least a activity of humanization of the present invention or its fragment antagonism Fc γ RIIB.In one embodiment of the invention, described activity is the receptor-mediated signal conduction of activating B cell.In a particular, humanization antagonistic antibodies of the present invention strengthens the activity that flows or strengthen one or more downstream signaling molecules in the Fc γ RIIB signal transduction path in B cell activity, the increment of B cell, antibody generation, the intracellular calcium.In the another one particular, phosphorylation or SHIP that humanization antagonistic antibodies of the present invention reduces Fc γ RIIB raise.In another one embodiment of the present invention, map kinase activity or Akt that the humanization antagonistic antibodies strengthens in the cell-mediated signal transduction path of B raise.In the another one embodiment, the inhibition to the conduction of Fc ε RI signal of humanization antagonistic antibodies antagonism Fc γ RIIB mediation of the present invention.In a particular, humanization antagonistic antibodies of the present invention strengthens the activation of Fc ε RI inductive mastocyte, calcium mobilization, takes off particle, cytokine generation or thrombotonin release.In the another one embodiment, humanization antagonistic antibodies of the present invention suppresses Fc γ RIIB phosphorylation, suppress SHIP raise, suppress the SHIP phosphorylation and with the activation that combines, strengthens map kinase family member (for example Erk1, Erk2, JNK, p38 etc.) of Shc.In the another one embodiment, humanization antagonistic antibodies of the present invention suppresses p62 ^DokTyrosine phosphorylation and with the combining of SHIP and rasGAP.In the another one embodiment, humanization antagonistic antibodies of the present invention strengthens the monocyte of Fc γ R mediation or the phagolysis of scavenger cell.In the another one embodiment, humanization antagonistic antibodies of the present invention prevents that the phagolysis of splenic macrophage, quilt from being nursed one's health particulate and removing.

Antibody of the present invention includes but not limited to: the antibody that monoclonal antibody, synthetic antibody, reorganization generate, multi-specificity antibody, human antibodies, chimeric antibody, camelization (camelized) antibody, strand Fvs (scFv), single-chain antibody, Fab fragment, F (ab ') fragment, disulphide connect the epitope binding fragment of Fvs (sdFv), intrabody (intradobies) and above-mentioned any antibody.Especially, the antibody that uses in the inventive method comprises the immunocompetence part of immunoglobulin molecules and immunoglobulin molecules, the molecule that promptly contains antigen-binding site, described antigen-binding site and Fc γ RIIB immunologic opsonin bonded avidity greater than with Fc γ RIIA bonded avidity.Antibody analog also can comprise Fc γ RIIB specific t-cell receptor, for example chimeric TXi Baoshouti (referring to, U.S. Patent Application Publication 2004/0043401), the single-chain T-cell receptor that is connected with single-chain antibody (referring to, U.S. Patent number 6,534,633) and the albumen support (referring to, U.S. Patent number 6,818,418).In certain embodiments, antibody analog of the present invention is not a monoclonal antibody.

The humanized antibody that the inventive method is used can be from any animal, comprise bird and Mammals (as, people, non-human primates, mouse, donkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken).Preferably, described antibody is human or humanized monoclonal antibody." mankind " used herein antibody comprises the antibody with human immunoglobulin aminoacid sequence, also comprises from the human immunoglobulin library or synthetic human immunoglobulin encoding sequence library or the antibody separated from the mouse of express human antibody.

The humanized antibody that uses in the inventive method can be monospecific, dual specific, tri-specific or polyspecific more.Multi-specificity antibody can immunologic opsonin in conjunction with different epitope on the Fc γ RIIB, perhaps immunologic opsonin is incorporated into epitope and the allos epi-position of Fc γ RIIB, as heterologous polypeptide or solid phase carrier material.Referring to: international publication number WO 93/17715, WO 92/08802, WO91/00360 and WO 92/05793; People such as Tutt, 1991, J.Immunol.147:60-69; U.S. Patent number 4,474,893,4,714,681,4,925,648,5,573,920 and 5,601,819; People such as Kostelny, 1992, JImmunol.148:1547-1553; People such as Todorovska, 2001 Journal of ImmunologicalMethods, 248:47-66.In specific embodiments, humanized antibody of the present invention is a polyspecific, specifically at Fc γ RIIB and cancer antigen or other for example in treatment or prevent the cell surface marker of certain cell that certain specified disease need be killed and wounded (for example immunocyte, as T cell or B cell); Perhaps specificity is at other Fc acceptor, for example Fc γ RIIIA, Fc γ RIIIB etc.

In a specific embodiments, the antibody that uses in the inventive method be a kind of antibody or its Fab (for example, comprise one or more complementary determining regions (CDR), be preferably whole 6 CDR), described antibody generates (for example heavy chain CDR3) by clone 2B6,3H7,1D5,2El, 2H9,2Dl1 or 1F2 (the ATCC preserving number is respectively PTA-4591, PTA-4592, PTA-5958, PTA-5961, PTA-5962, PTA-5960 and PTA-5959) respectively.In the another one embodiment, the antibody that uses in the inventive method and mouse monoclonal antibody are in conjunction with identical epitope, and described monoclonal antibody is generated by clone 2B6,3H7,1D5,2El, 2H9,2Dl1 or 1F2 (the ATCC preserving number is respectively PTA-4591, PTA-4592, PTA-5958, PTA-5961, PTA-5962, PTA-5960 and PTA-5959); And/or as in ELISA experiment or the experiment of other suitable competitive immunization, measure, antibody that uses in the inventive method and mouse monoclonal antibody competition, described antibody is generated by 2B6,3H7,1D5,2El, 2H9,2Dl1 or 1F2 (the ATCC preserving number is respectively PTA-4591, PTA-4592, PTA-5958, PTA-5961, PTA-5962, PTA-5960 and PTA-5959); And the used antibody of the inventive method combines with Fc γ RIIB, and avidity combines greater than described antibody or its Fab and Fc γ RIIA's.

The humanized antibody that uses in the inventive method comprises through the derivative of modifying, that is, and and by with any kind molecule and this antibody is covalently bound modifies.Such as but not limited to, these antibody derivatives comprise by the adorned antibody of following manner: by known protection/blocking groups, protein solubility cracking, be bonded to glycosylation, acetylize, Pegylation, phosphorylation, amidation, derivatize that modes such as cell aglucon or other albumen are carried out.Can carry out any amount of chemically modified by known technology, include but not limited to: the metabolism of specificity chemical cracking, acetylize, formylation, tunicamycin is synthetic etc.In addition, described derivative can comprise one or more nonclassical amino acids.

For some application of humanized antibody, be included in the application in interior application of human body or the vitro detection, preferred end user's antibody-like, chimeric antibody or humanized antibody.Human antibodies is completely used in special hope when the treatment human patients.Human antibodies can pass through the known prepared in various methods of prior art, comprises the phage display method of above-mentioned use from the antibody library of human immunoglobulin sequence.Referring to: U.S. Patent number 4,444,887 and 4,716,111; International publication number WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741; Wherein every piece of document is all quoted in full and is included in the present invention.

Can also use transgenic mice to generate human antibodies, the endogenous immunoglobulin that described mouse can not expressive function, but can express human immunoglobulin gene.For example, the immunoglobulin gene mixture of human heavy chain and light chain can be imported at random or homologous recombination is gone into the embryonic stem cell of mouse.Alternative, except human heavy chain and light chain gene, human variable region, constant region and region of variability also can be imported into the embryonic stem cell of mouse.Heavy chain of mouse and light chain immunoglobulin gene can be caused non-functional respectively or by the human immunoglobulin gene seat that homologous recombination is introduced simultaneously.Especially, J _HThe homozygous disappearance of functional domain has prevented the generation of endogenous antibody.Modified embryonic stem cell is bred and microinjection goes into to generate gomphosis mouse in the blastocyst.Gomphosis mouse is raised the homozygote generation for preparing express human antibody.Use ordinary method transgenic mice to be carried out immunity with the antigen of selecting (for example, polypeptide of the present invention is all or part of).Use conventional hybridization knurl technology, can be from through the transgenic mice of immunity, obtaining at above-mentioned antigenic monoclonal antibody.Human immunoglobulin transgenosis in the transgenic mice is reset in the B cell differentiation procedure, carries out class conversion and somatic mutation thereupon.Therefore, use may generate the technology that useful IgG, IgA, IgM and IgE antibody are gone up in treatment.The summary that is used to produce human antibodies about this technology, referring to: Lonberg and Huszar, 1995, Int.Rev.Immunol.13:65-93, the document is quoted in full includes the present invention in.Be used to produce human antibodies and human monoclonal antibody's the scheme that goes through and produce this antibody-like about this technology, referring to: international publication number WO 98/24893, WO 96/34096 and WO 96/33735; U.S. Patent number 5,413,923,5,625,126,5,633,425,5,569,825,5,661,016,5,545,806,5,814,318 and 5,939,598; All documents are quoted in full includes the present invention in.In addition, resemble Abgenix, Inc (Freemont, CA) and the such company of Medrax (Princeton NJ) can use as mentioned above the special needle of similar techniques to selected antigenic human antibodies.

Chimeric antibody is a kind of like this molecule, and wherein the different piece of antibody derives from different immunoglobulin molecules, for example has the antibody of non-human antibody variable region and human immunoglobulin constant region.The method of producing chimeric antibody is that prior art is known, referring to: Morrison, 1985, Science 229:1202; People such as Oi, 1986, BioTechniques 4:214; People such as Gillies, 1989, J.Immunol.Methods 125:191-202; U.S. Patent number 6,311,415,5,807,715,4,816,567 and 4,816,397; All documents are quoted in full includes the present invention in.The chimeric antibody that contains one or more non-human CDR and human immunoglobulin molecule framework region can use multiple known technology of the prior art to generate, and for example, CDR transplants (european patent number EP 239,400; International publication number WO 91/09967; With U.S. Patent number 5,225,539,5,530,101 and 5,585,089), frosting (veneering) or come to the surface again (resurfacing) (european patent number EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28 (4/5): 489-498; People such as Studnicka, 1994, Protein Engineering 7 (6): 805-814; With people such as Roguska, 1994, Proc.Natl.Acad.Sci.91:969), chain replaces (U.S. Patent number 5,565,332).The above-mentioned document of each piece is all quoted in full includes the present invention in.

In addition, use one of ordinary skill in the art known technology (referring to Greenspan ﹠amp; Bona, 1989, FASEBJ.1A31A44; Nissinoff, 1991, J.Immunol.147:2429-2438), antibody of the present invention can be used to generate anti-id AB conversely.The invention provides the method for utilizing polynucleotide, described polynucleotide comprises code book invention antibody or its segmental nucleotide sequence.

The present invention includes single functional zone antibody, the single functional zone antibody that comprises camelization is (referring to people such as Muyldermans, 2001, Trends Biochem.Sci.26:230; People such as Nuttall, 2000, Cur.Pharm.Biotech.1:253; Reichmann and Muyldermans, 1999, J.Immunol.Meth.231:25; International publication number WO 94/04678 and WO 94/25591; U.S. Patent number 6,005,079; All documents are quoted in full includes the present invention in).In one embodiment, the invention provides the single functional zone antibody that contains two VH districts, these two VH districts form single functional zone antibody through modifying.

Method of the present invention also comprises humanized antibody and segmental application thereof, this antibody or fragment Mammals preferably the transformation period in human body (for example plasma half-life) greater than 15 days, be preferably more than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months or greater than 5 months.The transformation period that humanized antibody of the present invention or its fragment increase in Mammals (being preferably the mankind) causes described antibody or antibody fragment higher blood plasma titre in this Mammals, thereby and has reduced the frequency of administration of described antibody or antibody fragment and/or reduced the concentration of institute's administration of antibodies or antibody fragment.Antibody or its fragment with interior transformation period of body of prolongation can generate by the known technology of one of ordinary skill in the art.For example, can prepare antibody or its fragment that the transformation period increases in the body by interactional amino-acid residue between modification (for example replace, lack or increase) participation Fc structural domain and the FcRn acceptor.Humanized antibody of the present invention can be transformed by the method that people such as Ward describe, to prolong biological half-life (referring to U.S. Patent number 6,277,375 B1).For example, humanized antibody of the present invention can be transformed at the Fc hinge area, with in the body with prolongation or plasma half-life.

Can be by polymer molecule (be attached to antibody or its fragment that antibody or its fragment prepare the transformation period in the body with prolongation as High molecular weight polyethylene glycol (PEG).Engage N or the C-terminal that PEG is connected to described antibody or antibody fragment by site-specific nature, perhaps by the ε amino on the lysine residue, PEG can be attached on the antibody or antibody fragment that has or do not have multifunction conjunction.Can use and cause minimum linearity or the branch-like polymer derivative of biological activity loss.To monitor yoke closely by SDS-PAGE and mass spectrometry and close degree, guarantee that the suitable yoke of PEG molecule and antibody closes.Unreacted PEG can separate with antibody-PEG mixture by for example size-exclusion or ion exchange chromatography.

Humanized antibody of the present invention can be modified (referring to U.S. Patent number 4,179,337) by method and coupling agent that people such as Davis describe, and purpose provides the composition that can be injected into the Mammals recycle system and not have immunogenic response substantially.

The present invention also comprises the application of humanized antibody or Fab, and described humanized antibody or Fab are included in the aminoacid sequence that there is any antibody of the present invention of variation (for example, one or more aminoacid replacement) in framework region or CDR zone.Preferably, the variation on these humanized antibodies is kept or has been strengthened affinity and/or the avidity of antibody to its immunologic opsonin bonded CD32B.Can measure the avidity of antibody with the known standard technique of one of ordinary skill in the art (as immunoassay) to specific antigen.

The present invention includes the modification of the framework region residue of humanized antibody of the present invention.Framework residue in the framework region can be replaced by the corresponding residue from CDR donor antibody, changes, preferably improves and combine with antigenic.These frameworks replace by the known method of prior art to be identified, for example, discern antigen in conjunction with important framework residue by the interaction model of making CDR and framework residue, by comparative sequences discern uncommon framework residue on the specific position (referring to, for example, U.S. Patent number 5,585,089; With people such as Riechmann, 1988, Nature 332:323; These documents are quoted in full includes the present invention in).

The present invention includes humanized antibody, this antibody contains modification, preferably contains the modification of change antibody and one or more Fc γ R avidity in the Fc zone.The bonded method that modified antibodies changes itself and one or more Fc γ R is that prior art is known, referring to: PCT publication number WO 04/029207, WO04/029092, WO 04/028564, WO 99/58572, WO 99/51642, WO 98/23289, WO89/07142, WO 88/07089 and U.S. Patent number 5,843,597 and 5,642,821, these documents are all quoted in full includes the present invention in.The present invention includes respectively disclosed any sudden change in the Application No. of submitting on January 9th, 2003 and on March 19th, 2,003 60/439,498 and 60/456,041, the mode that every piece of document is all quoted in full is included into the present invention.In some embodiments, the present invention includes the antibody that activation Fc γ R (as Fc γ RIIIA) avidity is changed.Preferably, this class is modified the effector function of the Fc mediation that also has change.The modification that influences the effector function of Fc mediation is prior art known (referring to U.S. Patent number 6,194,551, being included in the present invention to quote in full).Can be included but not limited to by modified amino acid according to the inventive method: proline 3 29, proline 3 31 and Methionin 322.Proline 3 29, proline 3 31 and Methionin 322 are preferably replaced by L-Ala, but also can consider to use other aminoacid replacement.Referring to: international publication number WO 00/42072 and U.S. Patent number 6,194,551, they are quoted in full includes the present invention in.

In a specific embodiments, the modification in Fc zone comprises one or more sudden changes in the Fc zone.One or more sudden changes in the Fc zone can produce reformed antibody, described change into antibody-mediated effector function, with the combining of other Fc acceptor (as the Fc activated receptor), ADCC activity, C1q in conjunction with cytotoxicity or phagolysis active, that complement relies on, the perhaps combination of these changes.

The antibody that the present invention also provides oligosaccharide content to change.Oligosaccharides used herein refers to contain the carbohydrate of two or more monose, and " oligosaccharides " and " carbohydrate " can exchange use at this paper.Carbohydrate part of the present invention will be named with reference to the term that generally uses in the prior art and describe.The summary of relevant carbohydrate chemistry, referring to: people such as Hubbard, 1981 Ann.Rev.Biochem, 50:555-583, way of reference is included into the present invention in full.This nomenclature for example comprises: Man represents seminose; GIcNAc represents the 2-N-acetylglucosamine; Gal represents semi-lactosi; Fuc represents trehalose; Glc represents glucose.Sialic acid represents that with contracted notation NeuNAc represents the 5-N-n acetylneuraminic acid n, and NeuNGc represents 5-glycerine neuraminic acid.

Usually, the carbohydrate residue is contained in the conservative position of heavy chain of antibody constant region, and nearly 30% IgG has glycosylated Fab district.IgG has two feelers (biantennary) carbohydrate structure that single N connects (people such as Jefferis, 1998, Immunol.Rev.163:59-76 in the Asn297 site that is positioned at the CH2 district; People such as Wright, 1997, Trends Biotech 15:26-32).IgG has following carbohydrate structure: GlcNAc (trehalose)-GlcNAc-Man-(ManGlcNAc) usually ₂But, the variation of carbohydrate content among the IgG takes place really, cause changing function, referring to: people such as Jassal, 2001 Biochem.Biophys.Res.Commun.288:243-9; People such as Groenink, 1996 J.Immunol.26:1404-7; People such as Boyd, 1995 MoI Immunol.32:1311-8; People such as Rumpel, 1994, Human AntibodyHybridomas, 5:143-51.Present invention resides in Asn297 and go up the reformed humanized antibody of bonded carbohydrate part.In one embodiment, the carbohydrate part has semi-lactosi and/or semi-lactosi-sialic acid on one or two terminal GlcNAc and/or the 3rd GlcNac arm (bisection GIcNAc).

In some embodiments, humanized antibody of the present invention does not contain the glycosyl group of one or more selections substantially, for example one or more sialic acid residueses, one or more galactose residue, one or more trehalose residue.Can use the known usual way vegetation of one of ordinary skill in the art not contain the antibody that one or more selected glycosyls are rolled into a ball substantially, comprise: for example, reorganization generates antibody of the present invention in host cell, described host cell is defectiveness in the glycosyl group process that the carbohydrate to antibody partly brings Selection In, like this glycosyl group of the selection that the antibody deficiency of 90-100% and carbohydrate partly link to each other in the composition.Other the selectable method for preparing this antibody-like comprises: for example, preventing or reducing culturing cell under the condition that adds one or more selected glycosyls group that perhaps one or more selected glycosyl group is removed in the translation back.

In a specific embodiments, the present invention includes the method for the basic homologous antibody preparation of preparation, wherein the carbohydrate of the antibody of about 80-100% partly lacks trehalose in the composition.Described antibody can prepare by the following method, and for example: (a) there is the host cell of the transformation of Trehalose Metabolism defective in use, and it carries out the ability drop of marine alga saccharification to expressed protein like this; (b) preventing or reducing culturing cell under the condition of trehalose acidylate; (c) trehalose is removed in the translation back, for example uses the mycoside enzyme; (d) antibody purification is chosen not by the product of trehalose acidylate.Most preferred, the nucleotides sequence of the required antibody of encoding is listed in expressed antibody is carried out expressing in the host cell that the ability of trehalose acidylate reduces.Preferably, host cell is that (lectin resistance CHO mutational cell line is referring to U.S. Patent Application Publication No. 2003/0115614 for Lee 13 Chinese hamster ovary celIs; PCT publication number WO 00/61739; European patent application EP 1 229 125; Ribka ﹠amp; Stanley, 1986, Somatic Cell ﹠amp; Molec.Gen.12 (1): 51-62; People such as Ripka, 1986 Arch.Biochem.Biophys.249 (2): 533-45), CHO-K1 cell, DUX-B11 cell, CHO-DP12 cell or CHO-DG44 cell, these cells are made antibody substantially not by the trehalose acidylate by modification.Therefore, described cell can have the trehalose transferring enzyme or add related other enzyme or expression of Substance and/or the active change of trehalose to the oligosaccharides that N connects, so that the activity of described enzyme in cell reduces or expression level descends.About producing the method for the antibody that content of trehalose changes, referring to: for example, WO03/035835; People such as Shields, 2002, J Bio1.Chem.277 (30): 26733-40; These two pieces of patent documentations way of reference in full are included into the present invention.

In some embodiments, reformed carbohydrate modification is adjusted following one or multinomial: the dissolving of antibody, promote transportation and antibody-secreting, the anti-assembling of promotion, conformation integrity and antibody-mediated effector function in the cell.In a specific embodiments, with respect to the antibody that does not have carbohydrate modification, the carbohydrate modification of change has strengthened antibody-mediated effector function.The carbohydrate modification that causes antibody-mediated effector function to change is well known in the prior art (referring to people such as Shields R.L., 2001, J.Biol Chem.277 (30): 26733-40; People such as Davies J., 2001, Biotechnology ﹠amp; Bioenginee

Ng, 74 (4): 288-294).In the another one specific embodiments, the carbohydrate modification of change has strengthened combining of antibody of the present invention and Fc γ RIIB acceptor.Change carbohydrate modification according to the inventive method and comprise, for example: strengthen the carbohydrate content of antibody or the carbohydrate content of reduction antibody.The method that changes carbohydrate content is that affiliated technical field personnel are known, referring to: people such as Wallick, 1988, Journal of Exp.Med.168 (3): 1099-1109; People such as Tao, 1989 Journal ofImmunology, 143 (8): 2595-2601; People such as Routledge, 1995 Transplantation, 60 (8): 847-53; People such as Elliott 2003; Nature Biotechnology, 21:414-21; People such as Shields 2002 Journal ofBiological Chemistry, 277 (30): 26733-40; All these documents are all quoted in full includes the present invention in.

In some embodiments, the present invention includes the humanized antibody that contains one or more glycosylation sites, one or more carbohydrate parts are covalently bound with this antibody.In the another one embodiment, present invention resides in the Fc district and contain one or more glycosylation sites and one or more modification, as the disclosed and one of ordinary skill in the art of preamble known those.In preferred embodiments, with respect to the antibody that contains wild-type Fc district, one or more modifications in Fc zone have strengthened the avidity of antibody to reactivity Fc γ R (for example, Fc γ RIIIA).The antibody of the present invention that contains one or more glycosylation sites and/or one or more modifications in the Fc district has the antibody-mediated effector function of enhanced, for example enhanced ADCC activity.In some embodiments, the present invention further comprises and contains one or more amino acid modified humanized antibodies, the known carbohydrate direct or indirect and antibody of adorned amino acid partly interacts, and includes but not limited to: be positioned at 241,243,244,245,245,249,256,258,260,262,264,265,296,299 and 301 amino acid.This interactional amino acid of carbohydrate direct or indirect and antibody is that prior art is known, referring to: people such as Jefferis, 1995 Immunology Letters, 44:111-7, the document is quoted in full includes the present invention in.

The present invention includes by one or more glycosylation sites are introduced the humanized antibody in one or more sites of antibody, preferably do not change the function of antibody, for example active with combining of Fc γ RIIB.Glycosylation site can be introduced into the variable region and/or the constant region of antibody of the present invention." glycosylation site " used herein comprises any concrete aminoacid sequence in the antibody, and specificity is covalently bound with it for oligosaccharides (carbohydrate that promptly contains two or more monose that connect together).Typically, the oligosaccharides side chain links to each other with the skeleton of antibody by N or C connection usually.The glycosylation that N-connects refers to that the oligosaccharides part is connected with the l-asparagine side chain.The glycosylation that O-connects refers to being connected of oligosaccharides part and hydroxyamino acid (for example Serine, Threonine).Antibody of the present invention can comprise one or more glycosylation sites, comprises the glycosylation site that is connected with O-that N-connects.Can use in the prior art any known N-of being used for to connect or O-connects glycosylated glycosylation site according to the present invention.According to the inventive method, the example that available N-connects glycosylation site is aminoacid sequence Asn-X-Thr/Ser, and wherein X can be any amino acid, and Thr/Ser represents Threonine or Serine.The known method in field was imported into antibody of the present invention under the present invention can be used in this class site.Referring to: " InVitro Mutagenesis, " Recombinant DNA:A Short Course, J.D.Watson waits the people, W.H.Freeman and Company, NewYork, 1983, chapter 8, and pp.106-116 is quoted in full and is included in the present invention.The method example that glycosylation site is imported antibody of the present invention can comprise the aminoacid sequence sudden change of modifying or inducing antibody, the Asn-X-Thr/Ser sequence that obtains to expect.

In some specific embodiments, the present invention includes the humanization Fc γ RIIB antibody of modification, wherein the total site Asn of the N-glycosylation in CDR2 district ₅₀-Val-Ser is modified, to remove the glycosylation site on the position 50.Though be not subjected to the restriction of any particular mechanism of action, the removal of glycosylation site can limit antibody generate in potential change and medicinal application in the potential immunogenicity.In a specific embodiments, the present invention includes a kind of humanized antibody, wherein the amino acid at 50 places, position is modified, for example, disappearance or replacement.In the another one specific embodiments, the present invention further is included in the amino acid modified of 51 places, position, for example lacks and replaces.In a specific embodiments, the present invention includes the humanized antibody that the amino acid of position 50 is replaced by tyrosine.In the another one embodiment, the amino acid that the present invention includes position 50 is by tyrosine replaces, the amino acid of position 51 is replaced by L-Ala humanized antibody.

In some specific embodiments, the present invention includes the method that changes the carbohydrate content of antibody of the present invention by increase or removal glycosylation site.The method that changes the antibody carbohydrate content is that prior art is known, and is included among the present invention, referring to U.S. Patent number 6,218,149; EP 0 359 096 B1; Application No. US 2002/0028486; WO 03/035835; US publication 2003/0115614; U.S. Patent number 6,218,149; Application No. 6,472,511; These documents are quoted in full includes the present invention in.In other embodiments, the present invention includes the method for partly adjusting carbohydrate content in the antibody by one or more endogenous carbohydrate of removing antibody.

The present invention further comprises the method for the effector function that changes antibody of the present invention, and this method comprises the carbohydrate content that uses the disclosed herein or known method of prior art to change antibody.

Can use the known standard technique of one of ordinary skill in the art in encoding antibody or its segmental nucleotide sequence, to introduce sudden change, comprise that for example: orthomutation and PCR mediated mutagenesis have caused aminoacid replacement.Preferably, with respect to original antibody or its fragment, derivative comprises: be less than 15 aminoacid replacement, be less than 10 aminoacid replacement, be less than 5 aminoacid replacement, be less than 4 aminoacid replacement, be less than 3 aminoacid replacement or be less than 2 aminoacid replacement.In a preferred embodiment, the nonessential amino-acid residue of one or more precognitions has been done conservative aminoacid substitutions.

The present invention also comprises humanized antibody or its fragment that contains variable heavy chain and/or variable light-chain amino acid sequence, at least 45% of the variable heavy chain of described aminoacid sequence and mouse monoclonal antibody and/or variable light-chain amino acid sequence, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% is identical, and described mouse monoclonal antibody is respectively PTA-4591 by the ATCC preserving number, PTA-4592, PTA-5958, PTA-5961, PTA-5962, the clone 2B6 of PTA-5960 and PTA-5959,3H7,1D5,2El, 2H9,2Dl1 or 1F2 generate.The present invention further comprise with Fc γ RIIB specificity bonded avidity greater than with antibody or its fragment of the avidity of Fc γ RIIA, described antibody or antibody fragment comprise the aminoacid sequence of one or more CDR, at least 45% of one or more cdr amino acid sequences of described aminoacid sequence and mouse monoclonal antibody, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% is identical, and described mouse monoclonal antibody is respectively PTA-4591 by the ATCC preserving number, PTA-4592, PTA-5958, PTA-5961, PTA-5962, the clone 2B6 of PTA-5960 and PTA-5959,3H7,1D5,2El, 2H9,2Dl1 or 1F2 generate.The homology per-cent of two aminoacid sequences can be measured by the known any method of one of ordinary skill in the art, comprises the retrieval of BLAST albumen.

The present invention also comprise with Fc γ RIIB specificity bonded avidity greater than the humanized antibody of Fc γ RIIA or the application of antibody fragment, the coding nucleotide sequence of wherein said antibody or antibody fragment and the nucleotides sequence of mouse monoclonal antibody are listed in to hybridize under the rigorous condition and form, and described mouse monoclonal antibody is generated by clone 2B6,3H7,1D5,2El, 2H9,2Dl1 or the 1F2 that the ATCC preserving number is respectively PTA-4591, PTA-4592, PTA-5958, PTA-5961, PTA-5962, PTA-5960 and PTA-5959.In a preferred embodiment, the invention provides and humanized antibody or the antibody fragment of Fc γ RIIB specificity bonded avidity greater than Fc γ RIIA, the nucleotides sequence of the variable light chain that described antibody or antibody fragment comprise and/or the coding nucleotide sequence of variable heavy chain and variable light chain of mouse monoclonal antibody and/or variable heavy chain is listed under the rigorous condition hybridizes, and described mouse monoclonal antibody is respectively PTA-4591 by the ATCC preserving number, PTA-4592, PTA-5958, PTA-5961, PTA-5962, the clone 2B6 of PTA-5960 and PTA-5959,3H7,1D5,2El, 2H9,2Dl1 or 1F2 generate.In the another one preferred embodiment, the invention provides and humanized antibody or the antibody fragment of Fc γ RIIB specificity bonded avidity greater than Fc γ RIIA, one or more CDR coding nucleotide sequences of the coding nucleotide sequence of one or more CDR that described antibody or antibody fragment comprise and mouse monoclonal antibody are hybridized under rigorous condition, and described mouse monoclonal antibody is respectively PTA-4591 by the ATCC preserving number, PTA-4592, PTA-5958, PTA-5961, PTA-5962, the clone 2B6 of PTA-5960 and PTA-5959,3H7,1D5,2El, 2H9,2Dl1 or 1F2 generate.Rigorous hybridization conditions includes but not limited to: hybridizing with the DNA that is fixed on the filtering membrane in 6 * sodium chloride/sodium citrate (SSC) under about 45 ℃, use 0.2 * SSC/0.1%SDS to wash one or many down then at about 50-60 ℃, highly rigorous condition for example, under about 45 ℃, in 6X SSC, hybridize with the DNA that is fixed on the filtering membrane, wash one or many with 0.1 * SSC/0.2%SDS down at about 60 ℃ then, known any other the rigorous hybridization conditions of perhaps affiliated technical field personnel (referring to: Ausubel, F.M. wait the people, 1989 Current Protocols in Molecular Biology, vol.1, Green Publishing Associates, Inc. with John Wiley and Sons, Inc., NY, 6.3.1-6.3.6 page or leaf and 2.10.3 page or leaf are included the present invention in way of reference).

5.1.1 antibody conjugates

The present invention includes humanized antibody and heterologous polypeptide (that is Wu Guan polypeptide; Or its part, being preferably at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acid of this polypeptide) reorganization is merged or chemical yoke closes (comprising that covalency and non-covalent yoke close) and generates fusion rotein.Merging must not be directly, can produce by joint sequence yet.By humanized antibody and specificity being merged at the antibody of specific cells surface receptor or yoke closes, this humanized antibody can for example be used for the heterologous polypeptide of in external or body target specific cells kind.Use the known method of prior art, also can be applied in external immunoassay and the purification process with the antibody that heterologous polypeptide merges or yoke closes, referring to: PCT publication number WO 93/21232; EP 439,095; People such as Naramura, 1994 Immunol.Lett., 39:91-99; U.S. Patent number 5,474,981; People such as Gillies, 1992 Proc.Natl.Acad.Sci.USA, 89:1428-1432; People such as Fell, 1991, J.Immunol, 146:2446-2452; These documents are quoted in full includes the present invention in.

Further, humanized antibody can close with the therapeutic preparation or the drug moiety yoke that change the particular organisms reaction.Therapeutic preparation or drug moiety are not limited to traditional chemotherapy preparation.For example, described drug moiety can be to have required bioactive protein or polypeptide.This proteinoid can comprise, for example: toxin such as abrin, ricin A, Pseudomonas exotoxin (being PE-40) or diphtheria toxin, Ricin, Bai Shusu (gelonin) and Pokeweed antiviral protein (pokeweed antiviral protein), protein is tumour necrosis factor for example, Interferon, rabbit (including but not limited to derive from the alpha-interferon (IFN-α) and the beta-interferon of somatomedin), nerve growth factor (NGF), the somatomedin (PDGF) in thrombocyte source, tissue plasminogen activator (TPA), the apoptosis agent (for example, TNF-α, TNF-β, disclosed AIMI among the PCT publication number WO 97/33899), AIMII (referring to: PCT publication number WO 97/34911), Fas part (people such as Takahashi, 1994J.Immunol, 6:1567-1574) and VEGI (PCT publication number WO99/23105), thrombosis agent or anti-angiogenic agent (for example angiostatin or endostatin), or biological response modifier such as lymph element (il-1 (" IL-1 ") for example, interleukin-2 (" IL-2 "), interleukin-6 (" IL-6 "), granulocyte phagocytic cell G CFS (" GM-CSF ") and granulocyte colony-stimulating factor (" G-CSF ")), macrophage colony stimulating factor (" M-CSF ") or somatomedin (for example tethelin (" GH ")); Proteolytic enzyme or rnase.

Humanized antibody can merge with flag sequence (as a kind of peptide), to make things convenient for purifying.In preferred embodiments, marker amino acid sequence is six polyhistidyls, and the label in the pQE carrier (QIAGEN, Inc., 9259 EtonAvenue, Chatsworth, CA, 91311) for example, other many carriers all can commercially obtain.For example, (1989 Proc.Natl.Acad.Sci.USA, 86:821-824) described six polyhistidyls have made things convenient for the purifying of fusion rotein as people such as Gentz.Other peptide tag that is used for purifying includes but not limited to: homo agglutinin " HA " label, corresponding to epitope (people such as Wilson from influenza hemagglutinin protein, 1984 Cell, 37:767) and " flag " label (people such as Knappik, 1994 Biotechniques, 17 (4): 754-761).

The present invention further comprises the composition that contains the heterologous polypeptide that closes with antibody fragment fusion or yoke.For example, heterologous polypeptide can with Fab fragment, Fd fragment, Fv fragment, F (ab) ₂Fragment or their part merge mutually or yoke closes.Polypeptide is merged mutually with antibody moiety or the bonded method is that prior art is known, referring to: U.S. Patent number 5,336,603,5,622,929,5,359,046,5,349,053,5,447,851 and 5,112,946; EP 307,434; EP 367,166; International publication number WO 96/04388 and WO 91/06570; People such as Ashkenazi, 1991, Proc.Natl Acad.Sci.USA 88:10535-10539; People such as Zheng, 1995, J.Immunol.154:5590-5600; People such as VII, 1992, Proc.Natl.Acad.Sci.USA 89:11337-11341 (above-mentioned document is quoted in full includes the present invention in).

The generation of other fusion rotein can be reset by gene rearrangement (gene-shuffling), motif, exon is reset and/or codon is reset (being referred to as " DNA rearrangement ") technology.DNA resets and can be used for changing antibody of the present invention or its segmental activity (for example, antibody or its fragment have higher avidity and lower dissociation rate).Generally referring to U.S. Patent number 5,605,793,5,811,238,5,830,721,5,834,252 and 5,837,458; People such as Patten, 1997, Curt.Opinion Biotechnol.8:724-33; Harayama, 1998, Trends Biotechnol.16:76; People such as Hansson, 1999, J.MoI Biol.287:265; Lorenzo and Blasco, 1998, Biotechniques 24:308 (these patents and publication are all quoted in full includes the present invention in).Before reorganization, insert or other method, can change antibody or its fragment or the antibody that is encoded or its fragment by random mutagenesis by error-prone PCR, random nucleotide.The polynucleotide of an encoding antibody or antibody fragment part or a plurality of parts (these parts combine with Fc γ RIIB specificity) can be with one of one or more heterologous molecule or polycomponent, motif, knot sections, partly, reorganization such as structural domain, fragment.

The present invention also comprises the humanized antibody that closes with diagnosis or therapeutic preparation or other molecule yoke, expects that increase its plasma half-life.As the part of Clinical Laboratory program, this humanized antibody can be used for the development or the progress of monitoring of diseases, illness or infection by diagnostic ground, judges the validity of given treatment plan.Can detect by antibody is combined conveniently with detectable substance.Detectable substance comprises: various enzymes, prothetic group, fluorescent substance, luminophore, noclilucence material, radioactive substance, positron emitting metal and inactive paramagnetic metal ion.Utilize known systems, can with detectable substance and the direct yoke of antibody closes or by intermediate (for example, the known linking agent of prior art) closes with the antibody indirect yoke, referring to: U.S. Patent number 4,741,900, metal ion wherein can close with the antibody yoke, can be used for diagnosis according to the present invention.The diagnosis of this class with detect can by with antibody and detectable substance mutually coupling finish, described detectable substance includes but not limited to: various enzymes include but not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase enzymes or acetylcholinesterase; The prothetic group mixture is such as but not limited to Streptavidin/vitamin H and avidin/vitamin H; Fluorescent substance includes but not limited to Umbelliferone, fluorescein, fluorescein isothiocyanate, rose-red, dichlorotriazine base amino (dichlorotriazinylamine) fluorescein, dansyl chloride or phycoerythrin; Luminophore is such as but not limited to luminol,3-aminophthalic acid cyclic hydrazide (luminol); The noclilucence material is such as but not limited to luciferase, luciferin and aequorin; Radioactive substance, such as but not limited to bismuth ( ²¹³Bi), carbon ( ¹⁴C), chromium ( ⁵¹Cr), cobalt ( ⁵⁷Co), fluorine ( ¹⁸F), gadolinium ( ¹⁵³Gd, ¹⁵⁹Gd), gallium ( ⁶⁸Ga, ⁶⁷Ga), germanium ( ⁶⁸Ge), holmium ( ¹⁶⁶Ho), indium ( ¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In), iodine ( ¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), lanthanum ( ¹⁴⁰La), lutetium ( ¹⁷⁷Lu), manganese ( ⁵⁴Mn), molybdenum ( ⁹⁹Mo), palladium ( ¹⁰³Pd), phosphorus ( ³²P), praseodymium ( ¹⁴²Pr), promethium ( ¹⁴⁹Pm), rhenium ( ¹⁸⁶Re, ¹⁸⁸Re), rhodium ( ¹⁰⁵Rh), ruthenium ( ⁹⁷Ru), samarium ( ¹⁵³Sm), scandium ( ⁴⁷Sc), selenium ( ⁷⁵Se), strontium ( ⁸⁵Sr), sulphur ( ³⁵S), technetium ( ⁹⁹Tc), thallium ( ²⁰¹Ti), tin ( ¹¹³Sn, ¹¹⁷Sn), tritium ( ³H), xenon ( ¹³³Xe), ytterbium ( ¹⁶⁹Yb, ¹⁷⁵Yb), yttrium ( ⁹⁰Y), zinc ( ⁶⁵Zn); The positron emitting metal and the on-radiation paramagnetic metal ion that are used for multiple positron emission tomography.

Antibody can close as cytotoxin (for example cytostatic agent or cytocide), therapeutic preparation or radioelement (as αFang Sheyuan, gamma ray radiator etc.) yoke with the therapeutic part.Cytotoxin or cellulotoxic preparation comprise the deleterious reagent of any pair cell, and the example comprises taxol, cytochalasin B, Gramicidin D, ethidium bromide, sphingosyl galactoside, mitomycin, Etoposide, vincristine(VCR), vinealeucoblastine(VLB), colchicine, Zorubicin, daunorubicin, dihydroxy anthracin, mitoxantrone, Plicamycin, dactinomycin, 1-boldenone, glucocorticosteroid, PROCAINE HCL, PHARMA GRADE, lignocaine, Proprasylyte, tetracycline and their analogue or homologue.Therapeutic preparation includes but not limited to: metabolic antagonist is (as methotrexate, Ismipur, 6-thioguanine, cytosine arabinoside, 5 FU 5 fluorouracil peace alkene miaow amine), alkylating agent is (as chlormethine, Chlorambucil (thioepa chlorambucil), melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, mitobronitol, streptozocin, ametycin and cisdichlorodiamine platinum (II) be cis-platinum (DDP)), anthrax cycline (as daunorubicin (predecessor is a daunomycin) and Zorubicin), microbiotic is (as dactinomycin (predecessor is an actinomycin), bleomycin, Plicamycin and Antramycin (AMC)), anti-fissuring agent (vincristine(VCR) and vinealeucoblastine(VLB)).

In addition, humanized antibody can close as the radioactive substance yoke with the therapeutic part, or closes with the macrocyclic chelants that is used to combine radiometal ion (referring to giving an example of above-mentioned radioactive substance) yoke.In certain embodiments, macrocyclic chelants is 1,4,7,10-tetraazacyclododecanand-N, N ', N ", " tetraacethyl (DOTA) can be connected on the antibody by linkers N.Described linkers is that prior art is known, referring to: people such as Denardo, 1998, Clin Cancer Res.4:2483-90; People such as Peterson, 1999, Bioconjug.Chem.10:553; People such as Zimmerman, 1999, Nucl.Med.Biol.26:943-50; These documents are quoted in full includes the present invention in.

Is known with above-mentioned therapeutic part with the technology that the antibody yoke closes, referring to: people such as Arnon, " Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy ", inMonoclonal Antibodies And Cancer Therapy, people such as Reisfeld (eds.), 1985, the 243-56 pages or leaves, Alan R.Liss, Inc.; People such as Hellstrom, " Antibodies For Drug Delivery ", inControlled Drug Delivery (the 2nd edition), people such as Robinson (eds.), 1987, the 623-53 pages or leaves, Marcel Dekker, Inc.; Thorpe, " Antibody Carriers Of Cytotoxic Agents In CancerTherapy:A Review ", in Monoclonal Antibodies ' 84:Biological And ClinicalApplications, people such as Pinchera (eds.), 1985, the 475-506 pages or leaves); " Analysis; Results; AndFuture Prospective Of The Therapeutic Use Of Radiolabeled Antibody In CancerTherapy ", in Monoclonal Antibodies For Cancer Detection 303-16 page or leaf, AcademicPress; People such as Thorpe, Immunol Rev., 62:119-58,1982.

Yoke closes or does not have yoke to close antibody or its fragment of therapeutic part, uses separately or co-administered with cytotoxic factor and/or cytokine, also can be used for the treatment of.

Alternative, antibody capable and second antibody yoke close and form antibody allos conjugates, as Segal described (U.S. Patent number 4,676,980, this patent are quoted in full includes the present invention in).

Antibody can also be connected on the solid phase carrier, and is particularly useful for the immunoassay or the purifying of target antigen.Described solid phase carrier includes but not limited to: glass, Mierocrystalline cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

5.2 the preparation of Fc γ RIIB humanized antibody

The present invention includes heavy chain that coding CDR transplants and light chain nucleotide sequence, contain the clonal expression carrier of described nucleotide sequence, with described nucleotide sequence transformed host cells and in transformed host cells, generating the method that contains this nucleotide sequence coded CDR transplanting chain and antibody molecule.In specific embodiments, the present invention includes any following nucleotide sequence: SEQ ID NOS.17,19,21,23,36 or 45.

The present invention includes the donor aminoacid sequence, this amino acid sequence encode and Fc γ RIIB bonded avidity greater than with Fc γ RIIA bonded antibody, as disclosed antibody in the following document: U.S. Provisional Patent Application number 60/403,366 (submission on August 14th, 2002) and U.S. Patent Application Publication No. 2004/0185045, these two pieces of documents are quoted in full includes the present invention in.In a specific embodiments, donor amino acid sequence encode monoclonal antibody, described monoclonal antibody is respectively PTA-4591 by the ATCC preserving number, PTA-4592, PTA-5958, PTA-5961, PTA-5962, the clone 2B6 of PTA-5960 and PTA-5959,3H7,1D5,2El, 2H9,2Dl1 or 1F2 generate, perhaps other monoclonal antibody that generates by immunization method of the present invention, as disclosed in the following document: U.S. Provisional Patent Application number 60/403,366 (submission on August 14th, 2002) and U.S. Patent Application Publication No. 2004/0185045, these two pieces of documents are quoted in full includes the present invention in.The present invention also comprises the polynucleotide of coding donor aminoacid sequence, this polynucleotide is (for example high rigorous degree under different rigorous conditions, in rigorous degree or low rigorous degree) with the polymerized nucleoside acid hybridization of coding monoclonal antibody, described monoclonal antibody is to be respectively PTA-4591 by the ATCC preserving number, PTA-4592, PTA-5958, PTA-5961, PTA-5962, the clone 2B6 of PTA-5960 and PTA-5959,3H7,1D5,2El, 2H9,2Dl1 or 1F2 generate, perhaps other monoclonal antibody that generates by immunization method of the present invention, as disclosed in the following document: U.S. Provisional Patent Application number 60/403,366 (submission on August 14th, 2002) and U.S. Patent Application Publication No. 2004/0185045.Hybridization can be carried out under the rigorous condition of difference.Such as but not limited to, the operating process under low rigorous condition following (referring to Shilo and Weinberg, 1981, Proc.Natl.Acad.Sci.U.S.A.78,6789-6792).The filter membrane that will contain DNA pre-treatment 6 hours in 40 ℃ solution, described solution contains 35% methane amide, 5 * SSC, 50 mM Tris-HCl (pH7.5), 5 mM EDTA, 0.1%PVP, 0.1% ficoll (Ficoll), 1%BSA and 500 μ g/ml sex change salmon sperm DNAs.Hybridization is carried out in same solution, and has done following change: 0.02%PVP, 0.02% ficoll (Ficoll), 0.2%BSA, 100 μ g/ml sex change salmon sperm DNAs, 10% (wt/vol) T 500, and uses 5-20 * 10 ⁶Cpm ³²The p-label probe.Filter membrane hybridization mixed solution in 40 ℃ hatched 18-20 hour, then in the solution that is containing 2 * SSC, 25mM Tris-HCl (pH 7.4), 5mM EDTA and 0.1%SDS under 55 ℃ the washing 1.5 hours.Change washing lotion with new solution, under 60 ℃, hatched again 1.5 hours.After being blotted, filter membrane carries out radioautograph.If necessary, filter membrane is washing for the third time under 65-68 ℃, takes pictures again then.Other operable low rigorous condition is the prior art known condition of species hybridization (as be used for).For example and not limitation, use the operating process of high rigorous condition as follows: the filter membrane that contains DNA under 65 ℃ in damping fluid prehybridization spent the night in 8 hours, described damping fluid consists of 6 * SSC, 50mMTris-HCl (pH 7.5), 1mM EDTA, 0.02%PVP, 0.02% ficoll, 0.02%BSA and 500 μ g/ml sex change salmon sperm DNAs; Filter membrane was being hybridized 48 hours in the prehybridization mixed solution under 65 ℃, and described prehybridization mixed solution contains 100 μ g/ml sex change salmon sperm DNA and 5-20 * 10 ⁶Cpm ³²The p-label probe.Filter membrane 37 ℃ with the solution washing that contains 2 * SSC, 0.01%PVP, 0.01% ficoll and 0.01%BSA 1 hour, radioautograph is carried out in washing 45 minutes in 0.1 * SSC under 50 ℃ subsequently afterwards.The high rigorous condition that other can adopt is well known in the prior art.The felicity condition of selecting rigorous degree is well known in the prior art (referring to people such as Sambrook, 1989, Molecular Cloning, the 2nd edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, New York; People such as Ausubel, inthe Current Protocols in Molecular Biology series of laboratory technique manuals, _ 1987-1997, Current Protocols, _ 1994-1997 John Wiley and Sons, Inc..With particular reference to: Dyson, 1991, " Immobilization of nucleic acids and hybridization analysis, " In:Essential Molecular Biology:A Practical Approach, Vol.2, T.A.Brown, ed., 111-156 page or leaf, IRL Press at Oxford University Press, Oxford, UK).Can obtain polynucleotide by any currently known methods of the prior art, and detect its nucleotide sequence.

The dna sequence dna of coding receptor aminoacid sequence can obtain by the known any method of one of ordinary skill in the art.For example, the encode dna sequence dna of preferred human receptor's framework sequence includes but not limited to FR fragment from human VH fragment VH1-8, JH6 and human VL fragment VK-A26, JK4.

In a specific embodiments, use conventional recombinant DNA technology that one or more CDR are inserted framework region.Described framework region is a framework region natural generation or total, is preferably human framework region (referring to people such as Chothia, 1998, J.MoI.Biol.278:457-479, human framework region tabulation).Preferably, a kind of antibody of polymerized nucleoside acid encoding that described framework region and CDR reorganization generate, described antibody and Fc γ RIIB specificity bonded avidity are greater than the avidity of described antibody and Fc γ RIIA.Preferably, as discussed above, can in framework region, carry out one or more aminoacid replacement, preferred described aminoacid replacement has strengthened combining of antibody of the present invention and Fc γ RIIB.

In the another one embodiment, can screen human library or other library of the prior art, clone the Nucleotide of those code book invention antibody by standard technique well known in the prior art.

Humanized antibody of the present invention can be generated by any method that is used to generate polypeptide well known in the prior art, for example, and synthetic in the body, recombinant DNA preparation etc.Preferably, humanized antibody is prepared by recombinant DNA technology.Humanization Fc γ RIIB specific antibody of the present invention can use the recombination immunoglobulin expression technology to generate.The reorganization of immunoglobulin molecules (comprising humanized antibody) is created in the following document open: U.S. Patent number 4,816,397 (people such as Boss), U.S. Patent number 6,331,415 and 4,816,567 (being all people such as Cabilly), English Patent 2,188,638 (people such as Winter) and English Patent GB 2,209,757; All documents are all quoted in full includes the present invention in.Immunoglobulin (Ig) (comprising Humanized immunoglobulin) recombination and expression techniques can also find in following document: people such as Goeddel, Gene Expression TechnologyMethods in Enzymology Vol.185 Academic Press (1991); Borreback, AntibodyEngineering, W.H.Freeman (1992).Out of Memory about generation, design and the expression of recombinant antibodies can find in following document: Mayforth, Designing Antibodies, Academic Press, SanDiego (1993).

The method example that is used for production recombinant humanized antibody of the present invention can comprise: the expression vector that a) makes up the operon that comprises the encoding antibody heavy chain by the molecular biology method of routine, the least part that wherein keeps required CDR of donor antibody binding specificity and variable region framework is from inhuman immunoglobulin (Ig), as mouse Fc γ RIIB monoclonal antibody, and the rest part of antibody is from human normal immunoglobulin, thereby generates a carrier that is used to express the humanization heavy chain of antibody; B) molecular biology method by routine makes up the expression vector of the operon that comprises the encoding antibody light chain, the least part that wherein keeps required CDR of donor antibody binding specificity and variable region framework is from inhuman immunoglobulin (Ig), Fc γ RIIB monoclonal antibody as mouse, and the rest part of antibody is from human normal immunoglobulin, thereby generates a carrier that is used to express the humanization light chain of antibody; C) change expression vector over to host cell by conventional molecular biology method and generate transfected host cell, be used to express the anti-Fc γ of humanization RIIB antibody; And d) cultivates transfected cell by conventional cell culture technology, so that produce the anti-Fc γ of humanization RIIB antibody.Host cell can be by two expression vector cotransfections of the present invention, and first carrier contains the derive operon of polypeptide of encoding heavy chain, and second carrier contains the operon of coding derived light chain polypeptide.Two carriers can contain different selected markers, but except heavy chain and light chain encoding sequence, two carriers are preferably identical.This process provides the equivalent of heavy chain and light chain polypeptide to express.Alternative, can use the single carrier of encoding heavy chain and light chain polypeptide simultaneously.The encoding sequence of heavy chain and light chain can comprise cDNA or genomic dna or both to be had.The host cell that is used for expressing recombinant antibodies of the present invention can be bacterial cell such as intestinal bacteria, perhaps is preferably eukaryotic cell.Preferably, can use mammalian cell, as Chinese hamster ovary cell or HEK-293.The selection of host cell is depended in the selection of expression vector, so that have the expression and the control characteristic of expectation in the host cell of selecting.Operable other clone includes but not limited to: and CHO-Kl, NSO and PER.C6 (Crucell, Leiden, Netherlands).

In a specific embodiments, the method that is used to generate humanization Fc γ RIIB 2B6 antibody comprises: the RNA from hybridoma cell line 2B6 is converted into cDNA, (Ambion Inc.) carries out pcr amplification to VH and VL fragment for example to use the RLM-RACE test kit.Used specific gene primer to VH.The example of VH primer comprises SJ15R, SEQ ID NO:47 (5 ' GGT CAC TGT CAC TGGCTC AGG G 3 ') and SJ16R, SEQ ID NO:48 (5 ' AGG CGG ATC CAG GGG CCAGTG GAT AGA C 3 '), the Auele Specific Primer of VL comprises SJ17R, SEQ ID NO:49 (5 ' GCACAC GAC TGA GGC ACC TCC AGA TG 3 ') and SJ18R, SEQ ID NO:50 (5 ' CGGCGG ATC CGA TGG ATA CAG TTG GTG CAG CAT C 3 ').(Invitrogen Inc.) inserts the RACE product in the plasmid (as pCR2.1-TOPO) to use TOPO TA clone test kit.Then the plasmid that generates is carried out dna sequencing, determine VH and the VL sequence of 2B6.Translation institute's calling sequence is also predicted the aminoacid sequence that each sequence determines.Defined as Kabat, from these sequences, identify framework (FR) and complementary determining region (CDR).Mouse VH is incorporated into people C-Gammal constant region and Ig homing sequence, and is inserted into pCI-neo and carries out the Mammals expression.Mouse VL is incorporated into people C-kappa fragment and Ig homing sequence, and also is cloned into pCI-neo and carries out the Mammals expression.Humanization 2B6 VH consists of: from the FR fragment of human VH fragment VH1-18 and JH6 and the CDR zone of 2B6 VH.Humanization 2B6 VL consists of: from the FR fragment of human VL fragment VK-A26 and JK4 and the CDR zone of 2B6 VL.Humanized VH and VL fragment begin assembling from the oligonucleotide of PCR combination and amplification.By PCR the fragment that generates is combined with homing sequence then, suitable constant region fragment is cloned into expression vector pCI-neo.Confirm the dna sequence dna of gained plasmid by sequential analysis.Afterwards, identification has the light chain segments of the human 2B6 VL sequence of prediction.Representational plasmid is that pMG * 608 (containing humanized 2B6 heavy chain) and pMGx611 (contain humanized 2B6 light chain, CDR2N ₅₀→ γ and V ₅₁→ A), be deposited in American type culture collection (10801 University Blvd. respectively on May 7th, 2004 according to being specified in of budapest treaty, Manassas, VA.20110-2209), the ATCC preserving number is respectively PTA-5963 and PTA-5964, and these two plasmids are included into the present invention with way of reference.

Construction of carrier of the present invention, usefulness generate the cell transfecting method of host cell of the present invention, the cell culture processes that usefulness generates antibody of the present invention, all are conventional molecular biology methods.Equally,, can adopt standard method of the prior art that it is carried out purifying, comprise cross flow filter, ammonium sulfate precipitation, affinity column chromatography, gel electrophoresis etc. in case recombinant humanized antibody of the present invention is generated.

Humanization Fc γ RIIB specific antibody of the present invention can be united or be connected in other antibody (or antibody moiety) and be used as human or humanized monoclonal antibody.These other antibody can react with other mark (epitope), and described mark is the characteristic antigen of antibody target disease of the present invention; Perhaps, described other antibody can have other characteristic, for example, diseased cells is arrived in the molecule or the cell aggregation of human immunity system.Antibody of the present invention (or its part) can with this antibody-like (or its part) Combined Preparation, can be the composition or a composition (chemical process or molecular biology method by routine connect both) of separate administration.In addition, can be by coming mark humanized antibody of the present invention with the label that can become the label of detectable signal or have a therapeutic property, thus improve the diagnosis and the therapeutic value of antibody of the present invention.Some labels as radionuclide, can generate detectable signal and have therapeutic property.The example of radionuclide includes but not limited to: ¹²⁵I, ¹³¹I and ¹⁴C.But other detection label comprises: fluorescent chromophore, as be used for fluorescein, phycobiliprotein or the tetraethylrhodamine that fluorescence microscopy is checked; Generate the enzyme of fluorescence or coloured product, be used for fluorescence, specific absorption, visible light or agglutination reaction and detect, perhaps this enzyme generates the high electron density product, shows by electron microscope technique; Perhaps a kind of high electron density molecule as ferritin, peroxidase or gold bead, is used for direct or indirect electron microscopic imaging.Mark with therapeutic property comprises the medicine that is used for the treatment of the cancer knurl, for example methotrexate etc.

The present invention is based on following discovery the humanized antibody of a lot of specificitys at Fc γ RIIB is provided: the CDR zone of mouse monoclonal antibody can be access in human receptor's framework and generate the humanization recombinant antibodies of specificity at Fc γ RIIB.Preferred humanization Fc γ RIIB specific antibody contains other at framework region (or other zone) and changes, to increase the combination to Fc γ RIIB.Particularly preferred embodiment of the present invention for example is the humanized antibody molecule that Fc γ RIIB is had super binding characteristic.

The present invention includes the standard recombinant dna method of the dna sequence dna that is used to prepare code book invention CDR grafted antibody.Can use oligonucleotide synthetic technology synthetic DNA sequence wholly or in part.The oligonucleotide directional synthesis method is well known in the prior art.The present invention further comprises site-directed mutafacient system, as those methods well known in the prior art.

Any suitable host/vector system may be used to express the dna sequence dna that coding CDR transplants heavy chain and light chain.Bacterium (as intestinal bacteria) and other microflora can be used in particular for the expressing antibodies fragment, for example Fab and (Fab ') ₂Fragment, especially FV fragment and single chain antibody fragments (as strand FVs).Eukaryotic system (as the mammalian host cell expression system) can be used to express the bigger CDR grafted antibody product that comprises complete antibody molecule.The mammalian host cell that is fit to comprises Chinese hamster ovary celI and myelomatosis or hybridoma cell line.Other spendable clone include but not limited to CHO-K1, NSO and PER.C6 (Crucell, Leiden, Netherlands).

Donor mouse antibodies of the present invention can use the known any method of prior art to generate, comprise following document disclosed method: U.S. Provisional Patent Application number 60/403,366 (submissions on August 14th, 2002) and 2004/0185045, two piece of document of U.S. Patent Application Publication No. are all quoted in full includes the present invention in.

The antibody fragment of identification specificity epitope can generate by known technology.For example, can use as papoid (generating the Fab fragment) or stomach en-(generation F (ab ') ₂Fragment) such enzyme, the proteolytic cleavage by immunoglobulin molecules generates Fab and (Fab ') ₂Fragment.F (ab ') ₂Fragment contains complete light chain and variable region, CHI district and at least a portion heavy chain hinge region.

For example, can use the known multiple phage display method of prior art to generate antibody.In the phage display method, the functional antibodies structural domain is illustrated in the phage particle surface of the polynucleotide that carries these antibody structure territories of coding.In a specific embodiments, can utilize such phage to show antigen binding domain, for example from Fab and the Fv or the stable Fv of disulfide linkage of antibody repertoire library or combinatorial antibody library (for example, people or mouse).Antigen expressed can the selected or identification by antigen in conjunction with the phage of territory (combining with target antigen), for example, applying marking antigen, perhaps combined or be trapped in the antigen of solid phase surface or globule.The phage of using in these methods is generally filobactivirus, comprises fd and M13.Antigen binding domain is expressed in phage gene III or the gene VIII albumen as recombination fusion protein.The example that can be used in preparation immunoglobulin (Ig) of the present invention or its segmental phage display method comprises that following document is disclosed: people such as Brinkman, J.Immunol.Methods, 182:41-50,1995; People such as Ames, J.Immunol.Methods, 184:177-186,1995; People such as Kettleborough, Eur.J.Immunol, 24:952-958,1994; People such as Persic, Gene, 187:9-18,1997; People such as Burton, Advances in Immunology, 57:191-280,1994; PCT application number PCT/GB91/01134; PCT publication number WO 90/02809, WO91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO95/20401 and U.S. Patent number 5,698,426,5,223,409,5,403,484,5,580,717,5,427,908,5,750,753,5,821,047,5,571,698,5,427,908,5,516,637,5,780,225,5,658,727,5,733,743 and 5,969,108; Every piece of document is all quoted in full includes the present invention in.

As describing in the above-mentioned document, after selecting phage, can be used to generate whole antibody (comprising the fragment that people's antibody or other are wanted) from the antibody coding region that phage is divided, and be expressed among any desired host, comprise mammalian cell, insect cell, vegetable cell, yeast and bacterium, as hereinafter describing in detail.For example, use, can also utilize reorganization to generate Fab, Fab ' and F (ab ') as disclosed currently known methods of the prior art in the following document ₂Segmental technology: PCT publication number WO 92/22324; People such as Mullinax, BioTechniques, 12 (6): 864-869,1992; People such as Sawai, AJRI, 34:26-34,1995; People such as Better, Science, 240:1041-1043,1988 (every piece of document is all quoted in full includes the present invention in).Can be used in the technical examples that generates strand Fv and antibody and comprise the technology of describing in the following document: U.S. Patent number 4,946,778 and 5,258,498; People such as Huston, Methods in Enzymology, 203:46-88,1991; People such as Shu, Proc.Natl.Acad.Sci.USA, 90:7995-7999,1993; People such as Skerra, Science, 240:1038-1040,1988.

Display technique of bacteriophage can be used in and strengthens the avidity of antibody of the present invention to Fc γ RIIB.This technology will be used for obtaining the high-affinity antibody that can use in combined method of the present invention.This technology (being called as affinity maturation) utilization mutagenesis or CDR walking (Walking) technology and selection again (with Fc γ RIIB or its antigenicity fragment discern with antigen-binding affinity than antibody original or that parental antibody is bigger) (referring to people such as Glaser, 1992, J.Immunology 149:3903).Whole codons (but not single Nucleotide) are carried out mutagenic treatment, form semirandom amino acid mutation spectrum.The library can be made up by various difference clones and form, and wherein the difference between each clone is that the single amino acids among the single CDR changes, and each clone contains the varient of each possibility aminoacid replacement of representing each CDR residue.By the fixed mutant is contacted with the antigen of mark, can screen the mutant that antigen avidity is increased.Can use the sudden change antibody that any known screening method in the prior art is discerned to be increased antigen avidity, as ELISA (referring to people such as Wu, 1998, Proc Natl Acad Sci.USA 95:6037; People such as YeWon, 1995, J.Immunology155:1994).Also can make the randomized CDR walking of light chain technology (referring to people such as Schier, 1996, J.MoIBio.263:551).

5.2.1 biological property is screened

Use well known in the prior art any based on the immunity or biochemical method (comprising that antagonist carries out quantitatively with the interaction of Fc γ RIIB) humanized antibody of the present invention is characterized with combining of Fc γ RIIB.Humanized antibody of the present invention combines with the specificity of Fc γ RIIB for example can utilize and measures based on immunity or biochemical method, includes but not limited to: ELISA, surface plasma resonance (surface plasmonresonance), immunoprecipitation, affinity chromatography, fluorescence-activated cell sorting (FACS) and equilibrium dialysis.The immunoassay that can be used in the immunologic opsonin combination of analyzing antibody of the present invention and cross reactivity includes but not limited to use the competitiveness and the noncompetitive detection architecture of following technology, as: western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), sandwich immunoassay, immune precipitation determination, lectin reaction, GDP reaction, immunodiffusion(ID) mensuration, agglutination reaction mensuration, complement be in conjunction with mensuration, immunoradiometric assay, fluorescence immunoassay, albumin A immunoassay, or the like.These assay methods all are that conventional, the known method of prior art is (referring to people such as Ausubel, eds, 1994, Current Protocolsin Molecular Biology, Vol.1, John Wiley ﹠amp; Sons, Inc., New York, the mode of being cited is included the present invention in).

Use external ELISA that humanized antibody of the present invention is characterized with combining of Fc γ RIIB.The example that the ELISA that the inventive method is used measures comprises: (this albumen is according to U.S. Provisional Application number 60/439 for the solubility Fc γ RIIb-Fc fusion rotein in 2.5ng/ hole, 709 and U.S. Patent number 10/756, the preparation of 153 disclosed methods, these two pieces of documents are quoted in full includes the present invention in) on 96 hole Maxisorp plates, caught room temperature 1 hour by mouse anti Fc γ RIIb antibody 3H7.Ch2B6 or hu2B6Hc/Ch2B6Lc conditioned medium begin to be added into each plate hole after a series of 2 times of dilutions from the concentration in 25ng/ hole.Orifice plate was at room temperature cultivated 1 hour, then by HRP link coupled F (ab ') ₂The anti-IgG F of goat (ab) ' ₂Specific secondary antibody detects combination.Hatch about 45 minutes with secondary antibody after, use TMB matrix to make dull and stereotyped colour developing.After hatching 5 minutes, use 1%H ₂SO ₄Termination reaction.Under OD450nm, read plate by the SOFTmax program.Between each step, wash orifice plate 3 times with the PBS/0.1% polysorbas20.Before adding solubility Fc γ RIIb-Fc, use the PBS/0.1% polysorbas20 that contains 0.5%BSA to dull and stereotyped room temperature sealing 30 minutes.

Use fluorescence activated cell sorting (FACS) and the known any technology of one of ordinary skill in the art that humanized antibody of the present invention is characterized with combining of Fc γ RIIB express cell (as Daudi cell and RajII cell).Flow sorter can a large amount of individual cells of quick test (as 10-100 1,000,000 cells/hour) (people such as Shapiro, Practical Flow Cytometry, 1995).The flow cytometer that is used for sorting and test organisms cell is well known in the prior art.Known flow cytometer is described in following document, for example: U.S. Patent number 4,347,935,5,464,581,5,483,469,5,602,039,5,643,796 and 6,211,477; These patents are all quoted in full includes the present invention in.The FACS Vantage that other known flow cytometer has Becton Dickinsonand Company to produce ^TMThe COPAS that system and Union Biometrica produce ^TMSystem.Be used to identify that the facs analysis example of humanized antibody of the present invention comprises: about 10 ⁶Individual Fc γ RIIB express cell (as Daudi cell and RajII cell) through damping fluid (as PBS) washing at least once.One-level antibody (as Ch2B6, Hu2B6Hc/ch2B6Lc, IgG 1) adds cell with being diluted into 0.5,0.1,0.02 μ g/mL among the PBS/1%BSA with 100 μ l dilution antibody.Cultivate down after 30 minutes for 4 ℃, once with 1ml PBS/1%BSA washed cell.The anti-human IgG Fc of PE link coupled goat specificity F (ab ') ₂(JacksonImmunoReseach Inc.) uses with dilution in 1: 1000 as secondary antibody fragment.Cultivate down after 30 minutes for 4 ℃, once with 1ml PBS/1%BSA washed cell.Cell suspension carries out facs analysis in the PBS/1%BSA of 500 μ l.Other clone that can be used for the inventive method includes but not limited to: through the CHO-Kl of CD32B transfection cell (hamster cell system); Through the CHO-Kl of CD32A transfection (hamster cell system); Through the 293H of CD32B transfection cell (human epithelial cell system); Through the 293H of CD32A transfection cell (human epithelial cell system); Raji cell (human Burkitt ' s lymphoma cell line); Daudi cell (human lymphoma clone) [Raji and Daudi B clone are only expressed endogenous CD32B]; Only express the THP-I cell (human monocyte system) of endogenous CD32A; Express the U937 (human monocyte system) of endogenous CD32A and CD32B; K526; HL60.

Humanized antibody of the present invention can make selecteed antibody have maximum specificity with respect to Fc γ RIIA to Fc γ RIIB by the epitope further illness that has not attacked the vital organs of the human body of mapping.The epitope mapping method of antibody is well known in the prior art, and is included within the method for the present invention.In certain embodiments, Fc γ RIIB or the fusion rotein that contains one or more Fc γ RIIB zone can be used for the epitope mapping to antibody of the present invention.In a specific embodiments, fusion rotein contains the aminoacid sequence in the Fc γ RIIB zone of merging mutually with the Fc part of IgG 2.The respective regions that each fusion rotein may further include with homoreceptor (as Fc γ RIIA) carries out aminoacid replacement and/or displacement to some zone of acceptor, and is as shown in table 2 below.PMGX125 and pMGX132 contain the IgG binding site of Fc γ RIIB acceptor, and the former has the C-terminal of Fc γ RIIB, and the latter has the C-terminal of Fc γ RIIA, can be used for distinguishing the C-terminal combination.Other one of N-terminal of IgG binding site and Fc γ RIIA or Fc γ RIIB have that Fc γ RIIA replaces and.These molecules can help to measure the part of acceptor molecule and antibodies.

Table 2 can be used to study the fusion rotein tabulation of the epitope of monoclonal anti Fc γ RIIB antibody.Residue 172-180 belongs to the IgG binding site of Fc γ RIIA and Fc γ RIIB.Amino acid from Fc γ RIIA sequence is represented with runic.

Plasmid	Acceptor	The N-end	172-180	SEQ ID NO：	The C-end	SEQ ID NO：
Plasmid	Acceptor	The N-end	172-180	SEQ ID NO：	The C-end	SEQ ID NO：	pMGX125	RIIb	IIb	KKFSRSDPN	51	APS-----SS(IIb)	57
pMGX126	RIIa/b	IIa	QKFSRLDPN	52	APS-----SS(IIb)	57	pMGX125	RIIb	IIb	KKFSRSDPN	51	APS-----SS(IIb)	57
pMGX126	RIIa/b	IIa	QKFSRLDPN	52	APS-----SS(IIb)	57	pMGX127		IIa	QKFSRLDPT	53	APS-----SS(IIb)	57
pMGX128		IIb	KKFSRLDPT	54	APS-----SS(IIb)	57	pMGX127		IIa	QKFSRLDPT	53	APS-----SS(IIb)	57
pMGX128		IIb	KKFSRLDPT	54	APS-----SS(IIb)	57	pMGX129		IIa	QKFSHLDPT	55	APS-----SS(IIb)	57
pMGX130		IIb	KKFSHLDPT	56	APS-----SS(IIb)	57	pMGX129		IIa	QKFSHLDPT	55	APS-----SS(IIb)	57
pMGX130		IIb	KKFSHLDPT	56	APS-----SS(IIb)	57	pMGX131		IIa	QKFSRLDPN	52	VPSMGSSS(IIa)	58
pMGX132		IIb	KKFSRSDPN	51	VPSMGSSS(IIa)	58	pMGX131		IIa	QKFSRLDPN	52	VPSMGSSS(IIa)	58
pMGX132		IIb	KKFSRSDPN	51	VPSMGSSS(IIa)	58	pMGX133	RIIa-131R	IIa	QKFSRLDPT	53	VPSMGSSS(IIa)	58
pMGX134	RIIb-131H	IIa	QKFSHLDPT	55	VPSMGSSS(IIa)	58	pMGX133	RIIa-131R	IIa	QKFSRLDPT	53	VPSMGSSS(IIa)	58
pMGX134	RIIb-131H	IIa	QKFSHLDPT	55	VPSMGSSS(IIa)	58	pMGX135		IIb	KKFSRLDPT	54	VPSMGSSS(IIa)	58
pMGX136		IIb	KKFSHLDPT	56	VPSMGSSS(IIa)	58	pMGX135		IIb	KKFSRLDPT	54	VPSMGSSS(IIa)	58

Can also use the known any analytical method of prior art to detect humanized antibody of the present invention, to characterize described antibody and the interactional kinetic parameter of Fc γ RIIB based on surface plasma body resonant vibration (SPR).Anyly can all can be used for the present invention by the commercial SPR instrument that obtains, include but not limited to: from BiacoreAB (Uppsala, BIAcore Instruments Sweden); By Affinity Sensors (Franklin, the IAsys instruments that MA.) provides; By Windsor Scientific Limited (Berks, the IBISsystem that UK) provides; By Nippon Laser and Electronics Lab (Hokkaido, the SPR-CELLIA system that Japan) provides; With by Texas Instruments (Dallas, the SPR Detector Spreeta that TX) provides.About summary based on the SPR technology, referring to: people such as Mullet, 2000, Methods 22:77-91; People such as Dong, 2002, Review in MoI.Biotech., 82:303-23; People such as Fivash, 1998, Current Opinion inBiotechnology 9:97-101; People such as Rich, 2000, Current Opinion in Biotechnology 11:54-61; All these documents are quoted in full includes the present invention in.In addition, be used to detect any SPR instrument of protein-protein interaction and also pay attention in the method for the invention based on the method for SPR, referring to: U.S. Patent number 6,373,577,6,289,286,5,322,798,5,341,215 and 6,268,125, all documents are quoted in full includes the present invention in.

In brief, relate to based on the assay method of SPR: will be fixed on the surface in conjunction with a right member, monitor in real time it with solution in should be between another right member interaction.The basis of SPR is the change that detects the solvent specific refractory power that takes place at described near surface when mixture forms and divide.The surface that is used for fixing is a sensor chip, is the core of SPR technology; Described sensor chip is made up of the glass surface of thin au by being coated with, and forms the substrate that design is used for optimizing the specific surfaces of molecule and surface bonding.Many kinds of sensor chips can commercially obtain, particularly from above-mentioned company buy or, all chips may be used in the method for the present invention.The example of sensor chip comprises the AB by BIAcore, the sensor chip of Inc. supply, for example CM5, SA, NTA and HPA.Molecule of the present invention can be fixed on the surface of sensor chip with any fixing means well known in the art and chemical process, this method comprises but does not limit by amino group directly covalently bound, directly covalently bound by sulfydryl, be connected with the vitamin H on the surface of avidin bag quilt, be connected with the aldehyde of carbohydrate group, and be connected with the NTA chip by histidine-tagged.

The present invention includes and characterize the antibody that the inventive method produced, it is used to identify the function of antibody of the present invention by use, and the characterization of particularly regulating Fc γ RIIB signaling activity carries out.For example, characterization of the present invention can detect the phosphorylation of the tyrosine residues in the ITIM motif of Fc γ RIIB, or detects the calcium mobilization's that B-cell receptor is produced inhibition.Characterization of the present invention can be based on cell or not based on the analysis of cell.

Commonly known in the art, in mastocyte, take off particle, calcium mobilization and cytokine that the copolymerization of the IgE acceptor Fc ε RI of Fc γ RIIB and high-affinity rally suppresses antigen induction generate (people 1997 such as Metcalfe D.D., Physiol.Rev.77:1033; Long E.O.1999 Annu Rev.Immunol 17:875).The molecular mechanism of this signal transduction path is being illustrated (OttV.L., 2002, J.Immunol.162 (9): 4430-9) in the recent period.In case with Fc ε RI copolymerization collection, the tyrosine site of Fc γ RIIB in the ITIM motif is by quick phosphorylation, raise the Src homologous region 2 (SHIP) (the SH2 structural domain that contains inositol polyphosphate-5-Phosphoric acid esterase) that contains inositol-5 Phosphoric acid esterase then, the Src homologous region subsequently by phosphorylation and with She and p62 ^DokIn conjunction with (p62 ^DokIt is the prototype of regulating molecule family, comprise signal conducting structure territory, the homeodomain of for example aminoterminal pleckstrin (pleckstrin) (PH structural domain), PTB structural domain and contain the PXXP motif and the C-terminal zone of a large amount of phosphorylation sites (people such as Carpino, 1997 Cell, 88:197; People such as Yamanshi, 1997, Cell, 88:205)).

The present invention includes and characterize of the adjusting of the anti-Fc γ of the present invention RIIB humanized antibody the reaction of one or more IgE mediations.Preferably, in characterizing the adjusting of antibody of the present invention, used the clone of coexpression IgE high-affinity receptor and Fc γ RIIB low-affinity receptor to the IgE mediated responses.In a specific embodiments, use the cell (RBL-H23 of the rat basophilic leukemia clone of whole person's class Fc γ RIIB transfection; People 1981 Eur.J.Immunol.11:317 such as Barsumian E.L. quote in full and include the present invention in) use in the method for the invention.RBL-2H3 is by the clear rat cell system that characterizes, and has been widely used the signal transduction mechanism behind the cell activation of studying the IgE mediation.When Fc γ RIIB expresses in the RBL-2H3 cell and during with Fc ε RI copolymerization collection, Fc γ RIIB suppressed Fc ε RI inductive calcium mobilization, take off particle and cytokine generates (people such as Malbec, 1998, J.Immunol.160:1647; People such as Oae τ on, 1995 J Clin.Invest.95:577; People such as Ott, 2002 J.of Immunol.168:4430-4439).

In some embodiments, the present invention includes sign anti-Fc γ RIIB humanized antibody of the present invention suppresses Fc ε RI inductive mastocyte activatory.For example, by the cell (RBL-H23 of the rat basophilic leukemia clone of Fc γ RIIB transfection; People 1981 Eut.J.Immunol.11:317 such as Barsumian E.L.) by IgE sensitization, and by the F of the anti-mouse IgG of rabbit (ab ') ₂Fragment stimulates assembles independent Fc ε RI, is perhaps stimulated by the anti-mouse IgG of complete rabbit and comes copolymerization collection Fc γ RIIB and Fc ε RI.In this system, by antibody of the present invention is added the described direct regulating effect of being analyzed the downstream signal transduction molecule in sensitization and the stimulated cells.For example, can analyze Fc γ RIIB tyrosine phosphorylation, SHIP raise activation, the p62 that includes but not limited to Erk1, Erk2, JNK or p38 with phosphorylation, map kinase family member ^DokTyrosine phosphorylation and it and the combining of SHIP and RasGAP.

Being used to detect antibody of the present invention comprises the exemplary assay method of Fc ε RI inductive mastocyte activatory restriction: with human Fc gamma RIIB transfection RBL-H23 cell; With IgE sensitization RBL-H23 cell; F (ab ') with the anti-mouse IgG of rabbit ₂Fragment stimulates the RBL-H23 cell (to assemble independent Fc ε RI and cause the signal conduction that Fc ε RI mediates, in contrast), perhaps stimulate RBL-H23 cell (come copolymerization collection Fc γ RIIB and Fc ε RI, produce inhibition) to the signal conduction of Fc ε RI mediation with the anti-mouse IgG of complete rabbit.Can also be with antibody preincubate of the present invention through the anti-mouse IgG antibody stimulated cells of complete rabbit.Detect with the cell of antibody preincubate of the present invention and do not rely on activity with the Fc ε RI of the cell of antibody preincubate of the present invention, and relatively the Fc ε RI in these cells relies on active level, can show that antibody of the present invention relies on active regulating effect to Fc ε RI.

Above-mentioned exemplary assay method can be used to identify antibody, these antibody blocking parts (IgG) and Fc γ RIIB receptors bind, and by preventing that Fc γ RIIB and Fc ε RI copolymerization collection from coming the inhibition to the conduction of Fc ε RI signal of antagonism Fc γ RIIB mediation.This assay method also can be identified other antibody, the copolymerization collection of Fc γ RIIB and Fc ε RI, and come the inhibition to the conduction of Fc ε RI signal of exciting Fc γ RIIB mediation by the copolymerization collection that promotes Fc γ RIIB and Fc ε RI.

In a preferred embodiment, Fc ε RI rely on activity be at least following one or more: regulate the downstream signal transduction molecule, as regulate Fc γ RIIB phosphorylation state, regulate SHIP and raise, regulate the map kinase activity, regulate the SHIP phosphorylation state, regulate SHIP and Shc in conjunction with SHIP and Shc, adjusting p62dok phosphorylation state, regulate p62dok and SHIP in conjunction with, regulate p62dok and RasGAP in conjunction with, regulate the calcium mobilization, regulate to take off particle and regulate cytokine and generate.In the another one preferred embodiment, it is that serotonin discharges and/or extracellular Ca that Fc ε RI relies on activity ⁺⁺The mastocyte activation that interior stream and/or IgE rely on.One of ordinary skill in the art are known: the copolymerization collection of Fc γ RIIB and Fc ε RI stimulates that Fc γ RIIB tyrosine phosphorylation, SHIP raise, SHIP tyrosine phosphorylation and SHIP combine with Shc's, and the copolymerization collection of Fc γ RIIB and Fc ε RI inhibition map kinase family member includes but not limited to the activation of Erk1, Erk2, JNK, p38.One of ordinary skill in the art know that also the copolymerization collection of Fc γ RIIB and Fc ε RI stimulates p62 ^DokTyrosine phosphorylation strengthens and it and the combining of SHIP and RasGAP.

In some embodiments, by the particle that takes off of monitoring and/or detection mastocyte or basophil, preferably in detection, characterize the ability that anti-Fc γ RIIB humanized antibody of the present invention is regulated the reaction of IgE mediation based on cell.Preferably, using the recombination method of the known standard of one of ordinary skill in the art will be used for the mastocyte of this detection or basophil is engineered to and contains human Fc gamma RIIB.In a specific embodiments, in analyzing, characterized the ability that the anti-Fc γ of the present invention RIIB humanized antibody is regulated the reaction of IgE mediation based on β-hexosaminidase (being included in the enzyme in the particle) release of cell, it is elementary event (people such as Aketani, 2001Immunol.Lett.75:185-9 acute allergy and the inflammatory conditions that β-hexosaminidase discharges from mastocyte and basophilic granulocyte; People such as Aketani, 2000 Anal.Chem.72:2653-8).The method according to this invention can detect the release of other inflammatory mediator, includes but not limited to that the release of serotonin and histamine detects replying of IgE mediation.Although be not subjected to the restriction of concrete mechanism of action, the particle that for example contains β-hexosaminidase from mastocyte and basophilic granulocyte release particles is the process that an intracellular calcium concentration relies on, and is started by the crosslinked of Fc ε RI and polyvalent antigen.

Be used to identify that the anti-Fc γ of the present invention RIIB humanized antibody is β-hexosaminidase release experiment to the exemplary assay method of the regulating effect of IgE mediated responses, comprise: with human Fc gamma RIIB transfection RBL-H23 cell; Separately with mouse IgE or mouse IgE and the common sensitization RBL-H23 cell of the anti-Fc γ of the present invention RIIB humanized antibody; Goat anti-mouse F (ab) with different concns ₂Stimulate the RBL-H23 cell, preferably 0.03 μ g/mL-30 μ g/mL stimulated 1 hour; Collect supernatant liquor; Lysing cell; Detect the activity that is released to the β-hexosaminidase in the supernatant liquor by colorimetry, for example use p-nitrophenyl N-acetyl-β-D-glucosamine glycosides to detect.Recently represent the activity of β-hexosaminidase of discharging to discharge active percentage with gross activity.Detect and the activity of the private antigen of competitive list, β-hexosaminidase of discharging in the cell with IgE, IgE and the anti-Fc γ of the present invention RIIB antibody treatment separately.Although be not subjected to the restriction of particular mechanism of action, in case cell is by the independent sensitization of mouse IgE and by the F of polyclone goat anti-mouse IgG (ab) ₂Fragment stimulates, and the gathering of Fc ε RI and crosslinked will take place, because the light chain of this polyclonal antibody identification and Fc ε RI bonded mouse IgE, thereby cause the activation of mastocyte and take off particle.On the other hand, when cell by mouse IgE and the anti-Fc γ of the present invention RIIB antibody sensitization together and by polyclone goat anti-mouse IgG F (ab) ₂When fragment stimulates, the crosslinked of Fc ε RI and Fc γ RIIB will take place, take off particle thereby suppress Fc ε RI inductive.In both cases, goat anti-mouse F (ab ') ₂Fragment all β-hexosaminidase of inductive dose dependence discharges.In some embodiments, do not influence the activation of inhibitory pathway, promptly under the situation in the presence of the anti-Fc γ RIIB antibody, take off particle level and do not change with Fc γ RIIB receptors bind and with the crosslinked anti-Fc γ RIIB antibody of Fc ε RI.In other embodiments, the stronger activation of the antibody-mediated inhibition acceptor of this anti-Fc γ RIIB (being Fc γ RIIB, when the time) with anti-Fc γ RIIB antibodies, thus form effectively crosslinked with Fc ε RI and activate the inhibitory pathway of homology accumulative Fc γ RIIB.

The present invention also comprises by the known calcium mobilization's detection method of one of ordinary skill in the art and characterizes the influence of the anti-Fc γ of the present invention RIIB humanized antibody to the cell response of IgE mediation.Exemplary calcium mobilization's detection method can comprise: with IgE initialize basophilic granulocyte or mastocyte; With calconcarboxylic acid such as Fura 2 incubated cells; Irritation cell as mentioned above; For example utilize the flow cytometry monitoring and/or measure intracellular calcium concentration.The present invention includes and use the known any method of one of ordinary skill in the art to monitor and/or measure intracellular calcium concentration.Referring to: Immunology Letters, 2001,75:185-9; British J.ofPharm, 2002,136:837-45; J of Immunology, 168:4430-9 and J.of Cell Biol, 153 (2): 339-49; All documents are all quoted in full includes the present invention in.

In preferred embodiments, anti-Fc γ RIIB humanized antibody of the present invention suppresses the cell activation of IgE mediation.In the other embodiment, Fc γ RIIB antibody blocking of the present invention is by the inhibitory pathway of Fc γ RIIB regulation and control or ligand-binding site point and the enhancing immunity reaction thus on the blocking-up Fc γ RIIB.

In some embodiments, express-measure if human mast cell has low endogenous Fc γ RIIB, then may be difficult to monitor and/or detect the difference in the antibody-mediated inhibitory pathway activation of the anti-Fc γ of the present invention RIIB as using the known standard method of prior art (as FACS dyeing).Therefore, the present invention includes other selectable method, can utilize cytokine and particular growth condition to raise Fc γ RIIB and express.There has been report Fc γ RIIB in following clone, highly to be raised: human monocyte system by IL4, as THP1 and U937 (people such as Tridandapani, 2002, J.Biol.Chem., 277 (7): 5082-5089) and primary human monocyte (people such as Pricop, 2001, J.of Immunol, 166:531-537).Reported the U937 cytodifferentiation that makes with dibutyryl ring AMP with the expression that increases Fc γ RII (people such as Cameron, 2002Immunology Letters 83,171-179).The expression of endogenous Fc γ RIIB in people's mastocyte that can utilize cytokine such as IL-4, IL-13 to raise to be used for the inventive method is measured sensitivity to increase.

The present invention also comprises the inhibition of the anti-Fc γ of sign humanization of the present invention RIIB antibody to the signal conduction of B-cell receptor (BCR) mediation.The signal conduction of BCR mediation can comprise at least one or a plurality of downstreams biologically, as activation and breeding, the antibody generation etc. of B cell.The copolymerization collection of Fc γ RIIB and BCR causes cell cycle progress and cell survival to be suppressed.In addition, the copolymerization collection of Fc γ RIIB and BCR causes the signal conduction of BCR mediation to be suppressed.

Especially, the signal conduction of BCR mediation comprises following at least a or multiple: regulate the downstream signal transduction molecule, for example the phosphorylation state of Fc γ RIIB, SHIP raise, location, map kinase activity, the AKt (anti-apoptotic signal) of Btk and/or PLC γ raise, calcium mobilization, cell cycle progress and cell proliferation.

Though many effector functions that the conduction of Fc γ RIIB mediation BCR signal suppresses mediate by SHIP, but prove recently, show calcium mobilization, the Ins (1 of significant Fc γ RIIB mediation from lipopolysaccharides (LPS) the activatory B cell of SHIP deficient mice, 4,5) P3 generates and the inhibition of Erk and Akt phosphorylation (people such as Brauweiler A., 2001, Journal of Immunology, 167 (1): 204-211).Therefore, the external B cell from the SHIP deficient mice can be used for characterizing antibody of the present invention.Measuring antibody of the present invention comprises the exemplary assay method that the BCR signal conduction of Fc γ RIIB mediation suppresses: separate spleen B cell from the SHIP deficient mice, activate described cell with lipopolysaccharides, with F (ab ') ₂Anti-IgM stimulates described cell to assemble BCR, perhaps stimulates described cell to come copolymerization collection BCR and Fc γ RIIB with anti-IgM.Stimulate and make BCR and Fc γ RIIB aggregating cells can be further and antibody preincubate of the present invention altogether with complete anti-IgM.Cell Fc γ RIIB relies on activity and can detect by standard technique well known in the prior art.Relatively rely on activity level, the antibody of the present invention that relatively will illustrate of these levels is relied on active regulating effect to Fc γ RIIB through the cell of antibody preincubate of the present invention with without the Fc γ RIIB in the cell of antibody preincubate of the present invention.

Detecting Fc γ RIIB dependence activity can comprise, for example mobilizes by flow cytometry detection intracellular Ca2+, detects the phosphorylation of Akt and/or Erk, detects PI (3,4,5) the P3 gathering that BCR mediates, and perhaps detects the B cell proliferation of Fc γ RIIB mediation.

The said determination method can be used for for example identifying antibody, this antibody is regulated the inhibition to the conduction of BCR signal that Fc γ RIIB mediates by part (IgG) binding site of blocking-up Fc γ RIIB acceptor, and by preventing that Fc γ RIIB and BCR from the inhibition to the conduction of BCR signal that the copolymerization collection comes antagonism Fc γ RIIB mediation taking place.Described assay method can also be used for identifying that the copolymerization collection of those promotion Fc γ RIIB and BCR and the BCR signal of exciting Fc γ RIIB mediation conduct the antibody that suppresses.

The present invention relates to characterize of the effect of the anti-Fc γ of humanization of the present invention RIIB antibody to the signal conduction of Fc γ RII mediation in human monocyte/scavenger cell.Utilize the SHIP action effect factor, the engulfing of the copolymerization collection downward modulation Fc γ R mediation of Fc γ RIIB and acceptor (people 2002 such as Tridandapani, J.Biol.Chem.277 (7): 5082-9) with immunoreceptor tyrosine-based activation motif (ITAM).The copolymerization collection of Fc γ RIIA and Fc γ RIIB makes the quick phosphorylation of tyrosine residues on the ITIM motif of Fc γ RIIB, thereby has promoted SHIP phosphorylation, SHIP to combine with Shc and molecular weight is 120 and the proteinic phosphorylation of 60-65 kDa.In addition, the copolymerization collection of Fc γ RIIA and Fc γ RIIB makes the downward modulation of Akt phosphorylation level, and Akt participates in cell to regulate and be responsible for suppressing apoptotic a kind of serine-threonine kinase.

The present invention further comprises the phagocytotic inhibition of the anti-Fc γ of sign humanization of the present invention RIIB antibody to Fc γ R mediation in human monocyte/scavenger cell.For example, can use the Fab fragment (Medarex of the mouse monoclonal antibody IV.3 of anti-Fc γ RIIA, Inc.) and goat anti-mouse antibody (assembling Fc γ RIIA separately) stimulate cell THP-1 from human monocyte system, perhaps use complete IV.3 mouse monoclonal antibody and goat anti-mouse antibody (coming copolymerization collection Fc γ RIIA and Fc γ RIIB) to stimulate cell THP-1 from human monocyte system.In this system, can be by antibody of the present invention be added the adjusting of measuring the downstream signal transduction molecule in stimulated cells, for example the combining of the tyrosine phosphorylation of Fc γ RIIB, SHIP phosphorylation, SHIP and Shc, Akt phosphorylation and molecular weight are 120 and the proteinic phosphorylation of 60-65 kDa.In addition, exist and lacking under the condition of antibody of the present invention, can directly detect the efficient of engulfing of monocytic Fc γ RIIB dependence.

Being used for measuring antibody of the present invention suppresses Fc γ R mediation in human monocyte/scavenger cell phagocytotic another exemplary detection method comprises: with mouse anti Fc γ RIIA antibody I Fab fragment (Medarex V.3, Inc.) and goat anti-mouse antibody (assemble Fc γ RIIA separately and start the signal conduction of Fc γ RIIA mediation) or use mouse anti Fc γ RIIA antibody and goat anti-mouse antibody (copolymerization collection Fc γ RIIA and Fc γ RIIB, and suppress the signal conduction of Fc γ RIIA mediation) stimulates the THP-I cell.Through mouse anti Fc γ RII antibody and goat anti-mouse antibody stimulated cells can also with antibody preincubate of the present invention.Detect and antibody preincubate of the present invention through irritation cell and with the Fc γ RIIA dependence activity in irritation cell of antibody preincubate of the present invention, and relatively the Fc γ RIIA in these cells relies on active level, and this will illustrate that antibody of the present invention relies on active regulating effect to Fc γ RIIA.

Described exemplary detection method can be used for identifying antibody, the part combination of this antibody blocking Fc γ RIIB acceptor, and the inhibition to the conduction of Fc γ RIIA signal of the antagonism Fc γ RIIB mediation by preventing Fc γ RIIB and Fc γ RIIA copolymerization collection.This detection method can also be used for identifying the inhibition to the conduction of Fc γ RIIA signal that those promote Fc γ RIIB and Fc γ RIIA copolymerization collection and exciting Fc γ RIIB mediation.

In another one specific embodiments of the present invention, the present invention relates to characterize the function of humanized antibody of the present invention, detect ability (the Tridandapani et ah of the sheep red blood cell (SRBC) (SRBC) of the fluorescently-labeled IgG conditioning of THP-1 cytophagy by preceding method, 2000, J.Biol.Chem.275:20480-7).For example, detecting phagocytotic illustrative methods comprises: with antibody of the present invention or do not handle the THP-1 cell with Fc γ RII bonded control antibodies; More described cell activity level, wherein (for example, the rosette activity is (with IgG for cytoactive; The THP-1 cell number that combines of SRBC of bag quilt), adhesive activity (with THP-1 cell bonded SRBC overall number) and phagocytic rate) difference will illustrate that antibody of the present invention relies on active regulating effect to Fc γ RIIA.This detection method can be used for identifying for example antibody, and the part combination of this antibody blocking Fc γ RIIB acceptor and antagonism Fc γ RIIB mediation are to phagocytotic inhibition.This assay method can also be identified the antibody to the inhibition of Fc γ RIIA signal conduction that those promote Fc γ RIIB mediation.

In a preferred embodiment, the Fc γ RIIB that humanized antibody of the present invention is regulated in the human monocyte/macrophage in following one or more modes relies on active: regulate that the downstream signal transduction molecule is for example regulated the phosphorylation state of Fc γ RIIB, regulated the SHIP phosphorylation, combine, regulate the Akt phosphorylation, adjusting molecular weight of regulating SHIP and Shc is about 120 and other proteinic phosphorylation of 60-65 kDa, and the adjusting phagolysis.

The present invention includes and use the known method of one of ordinary skill in the art to characterize the influence of humanized antibody of the present invention, for example, strengthen the active ability of tumour-specific ADCC of therapeutic antibodies the effector cell function of therapeutic antibodies.The therapeutic antibodies that can be used for the inventive method includes but not limited to anti-tumour antibody, antiviral antibody, antimicrobial antibody (as bacterium and unicellular parasite), its (5.3.6 joint) as described herein for example.Concrete, the present invention includes the effect of antibody of the present invention that characterize to the effector cell function of the therapeutic antibodies (as the tumour-specific monoclonal antibody) of Fc γ R mediation.The effector cell function that can analyze according to the present invention includes but not limited to: cytotoxicity, phagolysis, opsonization, conditioning phagocytosis, CIq combination and the cell-mediated cytotoxicity of complement-dependent of antibody dependent cellular mediation.Can use one of ordinary skill in the art known be used to measure the effector cell function active based on cell or not based on any assay method of cell (for the effector cell detection, referring to: people such as Perussia, 2000, Methods MoI Biol.121:179-92; People such as Baggiolini, 1998 Experientia, 44 (10): 841-8; People such as Lehmann, 2000J.Immunol Methods, 243 (1-2): 229-42; Brown EJ.1994, Methods Cell Biol, 45:147-64; People such as Munn, 1990 J.Exp.Med, 172:231-237; People such as Abdul-Majid, 2002 Scand.J.Immunol.55:70-81; People such as Ding, 1998, Immunity8:403-411, every piece of document is all quoted in full includes the present invention in).

Utilize the known standard method of one of ordinary skill in the art (referring to people such as Perussia, 2000, MethodsMoI Biol 121:179-92), can detect antibody of the present invention to therapeutic antibodies active influence of ADCC to Fc γ R mediation in effector cell (as natural killer cell)." cytotoxicity of antibody dependent cellular mediation " used herein and " ADCC " have the common and conventional sense of this area, refer to a kind of cell in vitro mediated responses, the non-specific cell poison cell of wherein expressing Fc γ R is (as monocyte, as natural killer cell (NK) and scavenger cell) discern bonded antibody on the target cell, thus make this target cell cracking.In principle, any effector cell with activation Fc γ R can both be activated and mediate ADCC.The main cell that is used to mediate ADCC is the NK cell of only expressing Fc γ RIII, and monocyte depends on that state of its activation, location or differentiation can express Fc γ RI, Fc γ RII and Fc γ RIII.The summary that relevant Fc γ R expresses on hematopoietic cell, referring to people such as Ravetch, 1991, Annu.Rev.Immunol., 9:457-92 is quoted in full and is included in the present invention.

The effector cell is the granulocyte of expressing one or more Fc γ R and exercising effector function.Preferably, this cell is expressed Fc γ RIII at least and is exercised the ADCC effector function.The effector cell that can be used for the inventive method includes but not limited to: peripheral blood lymphocytes (PBMC), natural killer cell (NK), monocyte and neutrophil leucocyte are preferably PBMC and NK.The effector cell can separate from its natural origin, separates from blood or PBMC as described herein.Preferably, the effector cell who is used for ADCC mensuration of the present invention is peripheral blood cell (PBMC), and described PBMC preferably uses for example Ficoll-Paque density gradient centrifugation purifying from normal blood of the known standard method of one of ordinary skill in the art.For example, whole blood is placed on the Ficoll-Paque layer, centrifugal 30 minutes kinds of 500g under the room temperature, PBMC can be separated.Collect leukocytic cream action effect daughter cell.Other effector cell that can be used for ADCC assay method of the present invention includes but not limited to the scavenger cell (MDM) of monocyte derived.The MDM that the action effect daughter cell uses in the inventive method perhaps uses fresh product (for example, from Advanced Biotechnologies, MD) preferably available from the freezing product.In most preferred embodiment, with the human monocyte of elutriation as the effector cell in the inventive method.The human monocyte of elutriation expresses activated receptors Fc γ RIIA, Fc γ RIIA and inhibition acceptor Fc γ RIIB.The human monocyte can commercial obtain, and also can obtain with the freezing form, and it thaws with the basic medium that contains the basic medium of 10% human AB blood plasma or contain the factor-containing human plasma.Fc γ R expression level can directly be measured in the cell, for example uses facs analysis.Selectable, cell also can develop into scavenger cell in nutrient solution.Fc γ RIIB expression level can be enhanced in the scavenger cell.The antibody that can be used to measure Fc γ R expression level includes but not limited to: anti-human Fc gamma RIIA antibody, as IV.3-FITC; Anti-Fc γ RI antibody is as 32.2 FITC; Anti-Fc γ RIIIA antibody is as 3G8-PE.

The target cell of using in the ADCC assay method of the present invention includes but not limited to: breast cancer cell line is the SK-BR-3 (referring to people such as for example Tremp, 1976, Cancer Res.33-41) of HTB-30 as the ATCC preserving number; The B-lymphocyte; Derived from the lymphocytic cell of Burkitts, as the ATCC preserving number is that the Raji cell of CCL-86 is (referring to people such as for example Epstein, 1965, J Nal Cancer Inst, 34:231-240), the ATCC preserving number is the Daudi cell (referring to people such as for example Klein, 1968, Cancer Res.28:1300-10) of CCL-213; Ovarian cancer cell line is that OVC AR-3 (referring to for example Hamilton, people such as Young, 1983), SK-OV-3, PA-I, CAOV3, OV-90 and the IGROV-I of HTB-161 (can be from NCI preservation center as the ATCC preserving number; People such as Benard, 1985, Cancer Research, 45:4970-9; Quoted in full and included in the present invention).Described target cell must be discerned by the antigen binding site of antibody to be measured.That the target cell of using in the inventive method can have is low, in or the cancer antigen presentation of high expression level.The cancer antigenic expression can use the known method of one of ordinary skill in the art to measure, as facs analysis.For example, the present invention includes the use ovarian cancer cell, as IGROV-I, wherein the expression level difference of Her2/neu, perhaps OV-CAR-3 (ATCC preserving number HTB-161 is characterized in that the Her2/neu expression level is lower than breast cancer cell line SK-BR-3).Other ovarian cancer cell line that can be used as the inventive method target cell comprises: and OVCAR-8 (people such as Hamilton, 1983, Cancer Res.43:5379-89 is quoted in full and includes the present invention in); SK-OV-3 (ATCC preserving number HTB-77); Caov-3 (ATCC preserving number HTB-75); PA-I (ATCC preserving number CRL-1572); OV-90 (ATCC preserving number CRL-11732); And OVCAR-4.Other breast cancer cell line that can be used for the inventive method comprises BT-549 (ATCC preserving number HTB-122), MCF7 (ATCC preserving number HTB-22) and Hs578T (ATCC preserving number HTB-126), and all are above-mentioned all can to obtain from NCI preservation center and ATCC.Other clone that can be used for the inventive method includes but not limited to: CCRF-CEM (leukemia); HL-60 (TB, leukemia); MOLT-4 (leukemia); RPMI-8226 (leukemia); SR (leukemia); A549 (nonsmall-cell lung cancer); EKVX (nonsmall-cell lung cancer); HOP-62 (nonsmall-cell lung cancer); HOP-92 (nonsmall-cell lung cancer); NC1-H226 (nonsmall-cell lung cancer); NC1-H23 (nonsmall-cell lung cancer); NC1-H322M (nonsmall-cell lung cancer); NC1-H460 (nonsmall-cell lung cancer); NC1-H522 (nonsmall-cell lung cancer); COLO 205 (Crohn disease); HCC-2998 (Crohn disease); HCT-116 (Crohn disease); HCT-15 (Crohn disease); HT29 (Crohn disease); KM 12 (Crohn disease); SW-620 (Crohn disease); SF-268 (CNS); SF-295 (CNS); SF-539 (CNS); SNB-19 (CNS); SNB-75 (CNS); U251 (CNS); LOX IMVl (melanoma); MALME-3M (melanoma); Ml 4 (melanoma); SK-MEL-2 (melanoma); SK-MEL-28 (melanoma); SK-MEL-5 (melanocyte look knurl); UACC-257 (melanoma); UACC-62 (melanoma); IGR-OVl (ovarian cancer); OVCAR-3,4,5,8 (ovarian cancers); SK-OV-3 (ovarian cancer); 786-0 (kidney); A498 (kidney); ACHN (kidney); CAKl-I (kidney); SN12C (kidney); TK-IO (kidney); UO-31 (kidney); PC-3C (prostate cancer); DU-145 (prostate cancer); NC1/ADR-RES (mammary cancer); MDA-MB-231/ATCC (mammary cancer); MDA-MB-435 (mammary cancer); DMS 114 (small cell lung cancer); And SHP-77 (small cell lung cancer); Above-mentioned all cells all can obtain from NCL.

A kind ofly detect antibody of the present invention the illustrative methods of therapeutic antibodies ADCC activity influence is based on ⁵¹Cr discharges mensuration, comprising: with [ ⁵¹Cr] Na ₂CrO ₄(but molecule that should permeate through cell membranes is generally to be used for the molecule of mark, because it combines with cytoplasm protein, although and its spontaneously slowly release from cell, mainly after the target cell cracking, discharge in a large number) the labels targets cell, preferably, target cell is expressed one or more tumour antigens; Antibody of the present invention (for example 2B6,3H7) exist and non-existent situation under, nurse one's health target cell with one or more with the antibody that the tumour antigen of expressing on the target cell surface combines; To in titer plate, contact in the proper ratio with the effector cell through the radiolabeled target cell of conditioning; The incubated cell mixture was preferably hatched under 37 ℃ 16-18 hour; Collect supernatant liquor; Analyze the radioactivity in the supernatant samples then.Existing and not existing under the antibody condition of the present invention, measure the cytotoxicity of therapeutic antibodies, for example use following formula: specificity cracking per-cent=(experimental cracking-antibody dependent/non-dependent cracking/maximum cracking-antibody dependent/non-dependent cracking) * 100%.Generate graphic representation by ratio or the antibody concentration that changes target cell and effector cell.

In the another one embodiment, according to the influence of preceding method sign antibody antagonist dependent cell toxic action of the present invention (ADCC), referring to people such as Ding, Immunity, 1998,8:403-11, it is quoted in full includes the present invention in.

In some embodiments, the present invention includes sign antibody of the present invention and in experiment in vitro and/or animal model, strengthen the active function of therapeutic antibodies ADCC.

In a specific embodiments, the present invention includes and use ovarian cancer model and/or breast cancer model to characterize the function that humanized antibody of the present invention strengthens tumour-specific ADCC.

Preferably, ADCC of the present invention analyzes and used more than a kind of cancerous cell line, the feature of described clone is to have a kind of cancer antigen presentation at least, and wherein the cancer antigenic expression is different in the cancerous cell line that uses.Although be not intended to be subjected to the restriction of particular mechanism of action, in the different clone of multiple cancer antigenic expression, carry out ADCC and detect the preciseness that to measure cleaning antibody tumour of the present invention.In one embodiment, ADCC of the present invention detects and is to use cancerous cell line with different carcinoma antigenic expression to carry out.

In an exemplary detection, OVCAR3 (a kind of ovarian cancer cell line) can be used as expressing tumor antigen Her2/neu and TAG-72 tumour target; The human monocyte who expresses activatory Fc γ RIIIA and Fc γ RIIA and inhibition Fc γ RIIB can be used as the effector cell; Tumour-specific mouse antibodies ch4D5 and chCC49 can be used as tumor specific antibody.OVCAR3 can obtain from ATCC (preserving number HTB-161).Preferably, in the substratum that has added the 0.01mg/ml Sigma I8405, cultivate the OVCAR-3 cell.Can be with 5 * 10 ⁶Great-hearted OVCAR-3 cell is subcutaneously injected in the athymic nude mice of age and body weight coupling with Matrigel (Becton Dickinson).Can calculate the tumor weight of estimating by following formula: length-(width) ²/ 2, preferably be no more than 3g.6-8 separates grappling dependent tumors (anchorage-dependent tumor) after week, adding 1 μ g Collagenase (Sigma) and 5mg/mL RNA enzyme disperse cell in every gram tumour, come isolated cell by cell filter and nylon filtering net.Cell is frozen prolonged preservation subsequently, is used for subcutaneous injection and sets up xenograft models.

The hybridoma of secretion CC49 and 4D5 antibody can obtain (preserving number is HB-9459 and CRL-3D463) from ATCC, and its heavy chain and light-chain amino acid sequence are that known (referring to people such as Murray, 1994Cancer 73 (35): 1057-66; People such as Yamamoto, 1986 Nature, 319:230-4; These two pieces of documents are quoted in full includes the present invention in).Preferably, yes use chimeric 4D5 of the known standard method of one of ordinary skill in the art and CC49 antibody, thus make human Fc sequence (for example IgG 1 constant region) by grafting in the variable region of mouse antibodies so that effector function to be provided.Chimeric CC49 and 4D5 antibody bind their Fc district of merga pass by their variable region and target cell and go up the Fc γ R that expresses with human effector cell and combine.CC49 is at TAG-72, and it is high-molecular weight mucins (people such as Lottich, 1985 Breast Cancer Res.Treat.6 (1): the 49-56 of high level expression on many adenocarcinoma cells and ovarian cancer cell; People such as Mansi, 1989 Int.J.Rod.Appl Instrum are (2): 127-35 B.16; People such as Colcher, 1991 Int.J.Rad.Appl.Instrum are B.18:395-41; All documents are quoted in full includes the present invention in).4D5 is at human epidermal growth factor acceptor 2 (people such as Carter, 1992, Proc.Natl.Acad.Sci.USA, 89:4285-9, it is quoted in full includes the present invention in).Antibody of the present invention can be used to study the ADCC increased activity of tumor specific antibody by blocking-up inhibition Fc γ RIIB.Although be not subjected to the restriction of particular mechanism of action, activation along with the effector cell of expressing at least a excitability Fc γ R (as Fc γ RIIA), the expression of inhibition acceptor (Fc γ RIIB) will increase, and this has limited the removing of tumour, because the ADCC activity of Fc γ RIIA has been subjected to inhibition.But antibody of the present invention can promptly prevent the antibody that the inhibition signal is activated as blocking antibody, thereby activation signals (as the ADCC activity) will be kept the longer time and can remove tumour effectively.

Preferably, be used to strengthen the active humanized antibody of the present invention of ADCC and contained by modification that at least one is amino acid modified, thereby their Fc district is weakened with combining of Fc γ R, most preferably eliminate.In some embodiments, antibody of the present invention is contained by modification that at least one is amino acid modified, compare with wild-type antibody of the present invention, reduced combining of constant region and excitability Fc γ R (as Fc γ RIIIA, Fc γ RIIA), it is active to have kept maximum Fc γ RIIB blocking-up simultaneously.Can modify antibody of the present invention according to the known or disclosed by the invention any method of one of ordinary skill in the art.Any amino acid modified can being used in the method for the present invention of known energy damage effect subfunction, disclosed in for example following document: Application No. 60/439,498 (submissions on January 9th, 2003) and 60/456,041 (submission on March 19th, 2003), these two pieces of documents are quoted in full includes the present invention in.In some embodiments, antibody of the present invention is modified in 265 sites, and for example, 265 are replaced by L-Ala in the position.In preferred embodiments, the mouse constant region in the antibody of the present invention is by corresponding human constant region displacement, and this human constant region is replaced by L-Ala at the amino acid in 265 sites, thereby has eliminated effector function and to have kept Fc γ RIIB blocking-up active.ELISA detects and shows, the single amino acid that carries out in IgG1 heavy chain 265 sites changes and can significantly reduce and the combining of Fc γ R, and causes gross tumor volume to reduce (people such as Jefferis, 1995, Immunol Lett44:111-117 is quoted in full and is included in the present invention).In other embodiments, antibody of the present invention is modified in 297 sites, and for example position 297 is replaced by glutamine, thus eliminated the glycosylation site that N connects (referring to people such as Jejferies, 1995, Immunol, left 44:111-7; People such as Lund, 1996, J.Immunol, 157:4963-69; People such as Wright, 1994, J Exp.Med.180:1087-96; People such as White, 1997, J.Immunol.158:426-35; All documents are quoted in full includes the present invention in).The modification of having reported this site can be eliminated the interaction of all and Fc γ R.In preferred embodiments, the mouse constant region of antibody of the present invention is by corresponding human constant region exchange, and the amino acid in 265 and/or 297 sites of this constant region is substituted, thereby has eliminated effector function and to have kept Fc γ RIIB blocking-up active.

Exist or do not exist under the antibody condition of the present invention, the active illustrative methods of ADCC that detects tumor specific antibody is based on on-radiation europium (BATDA, Perkin Elmer) fluoroscopic examination, can comprise: strengthen ester acyl-oxygen methyl ester (acteoxylmethyl ester) with fluorescence and come the labels targets cell, this ester forms wetting ability part (TDA) (this mixture can not break away from cell, and only just can be released) by hydrolysis and cytolemma after effector causes lysis; Target cell with mark under anti-tumour antibody and antibody existence condition of the present invention joins in the effector cell; Hatched target cell and effector cell mixture 6-16 hour, and preferably hatched at 37 ℃.By detection be released and with europium (DELFIA reaction reagent; PerkinElmer) Fan Ying amount of ligand is measured the activity of ADCC.This part and europium form highly stable high fluorescence huge legendary turtle compound (EuTDA), and detected fluorescence is directly proportional with the number of lysing cell.Can use following formula to calculate specificity cracking per-cent: (experimental cracking-antibody dependent/non-dependent cracking/maximum cracking-antibody dependent/non-dependent cracking) * 100%.

In some embodiments, if too low,, then the present invention includes use and detect, for example based on radioactive ADCC to such an extent as to can not detect the ADCC activity of therapeutic antibodies based on the ADCC detection sensitivity of fluorescence ⁵¹Cr discharges mensuration.Can substitute based on the ADCC of fluorescence based on radioactive detection and to detect, perhaps detect the associating use with ADCC based on fluorescence.

Be used to characterize the exemplary of antibody of the present invention ⁵¹Cr discharges detection method and can comprise: use ⁵¹Cr mark 1-2 * 10 ⁶Target cell (as the OVCAR-3 cell); Under the condition that has and do not exist antibody of the present invention, nurse one's health target cell, and add 5 * 10 with antibody 4D5 and CC49 ³Cell is (preferred, the concentration range of 4D5 and CC49 is 1-15 μ g/mL) in 96 orifice plates; With the scavenger cell (MDM) (effector cell) of the adding of the target cell after conditioning monocyte derived, preferred proportional range is 10: 1-100: 1; Cell mixture was hatched under 37 ℃ 16-18 hour; Collect supernatant liquor; Analyze the radioactivity in the supernatant samples then.Exist and do not exist under the condition of antibody of the present invention, can measure the cytotoxicity of 4D5 and CC49, for example adopt following formula to determine: specificity cracking per-cent=(experimental cracking-antibody dependent/non-dependent cracking/maximum cracking-antibody dependent/non-dependent cracking) * 100%.

In some embodiments, the activity in vivo of Fc γ RIIB humanized antibody of the present invention is measured in heterograft human tumor model.Can use above-mentioned any cancerous cell line to make up tumour.In some embodiments, make up tumour with two kinds of cancerous cell lines, wherein the feature of first cancerous cell line is a low expression level cancer antigen, and the feature of second cancerous cell line is the identical cancer antigen of high level expression.Then, use the known method of one of ordinary skill in the art, the cancer antigen immune specificity bonded anti-tumour antibody of the utilization and first and second cancerous cell lines and suitable mouse model such as Balb/c nude mice model are (as Jackson Laboratories, Taconic), the person monocytic cell of suitable transfection and MDM action effect daughter cell are measured the tumour removing.Can in this animal model, detect above-mentioned any antibody to estimate the effect of the anti-Fc γ of the present invention RIIB antibody in tumour is removed.Can be used for mouse of the present invention and comprise, for example: Fc γ RIII-/-(wherein Fc γ RIIIA is knocked out); Fc γ-/-nude mice (wherein Fc γ RI and Fc γ RIIIA are knocked out); Perhaps human Fc gamma RIIB knocks in (knock in) mouse or genetically modifiedly knocks in mouse, and wherein fcgr2 on the mouse chromosome 1 and fcgr3 locus are expressed human Fc gamma RIIA, human Fc gamma RIIA, human Fc gamma RIIB, human Fc gamma RIIC, human Fc gamma RIIIA and Fc γ RIIIB by inactivation and mouse.

The illustrative methods that is used to detect antibody activity in vivo of the present invention can comprise: use to express cancer antigen and make up the heterograft mouse model as the cancerous cell line of feature, and measure antibody of the present invention to specificity at the influence of the antigenic antibody of the cancer of expressing in the cancerous cell line in the mediation tumour is removed.Preferably, use two kinds of above-mentioned cells in vivo activity of the parallel detection of cancerous cell line, wherein, the characteristics of first cancerous cell line are the first cancer antigen of low expression level, and the characteristics of second cancerous cell line are the identical cancer antigen of high level expression (with respect to first cancerous cell line).These experiments will strengthen the preciseness to antibody of the present invention evaluation of role in tumour is removed.For example, can make up tumour and estimate the effect of the anti-Fc γ of the present invention RIIB antibody in the tumour of Her2/neu specific antibody is removed with IGROV-I clone.In order to set up the heterograft tumor model, can be with 5 * 10 ⁶Great-hearted cell (as IGROV-I, SKBR3) injection for example uses Matrigel (BectonDickinson) subcutaneous injection to give mouse (the female nude mice that is complementary as 8 ages and body weight).The tumor weight of estimating can be determined by following formula: length * (width) ²/ 2, be preferably and be no more than 3 grams.The subcutaneous injection of IGROV-I cell produces quick growing tumors, and the intraperitoneal approach is induced peritoneal cancer diffusion, can kill mouse (people such as Benard, 1985, Cancer Res..45:4970-9) in 2 months.Because the IGROV-I cell forms tumour in 5 weeks, therefore used back first day at tumour cell, monocyte action effect daughter cell is carried out intraperitoneal with specificity at the therapeutic antibodies (as Ch4D5) of Her2/neu and antibody of the present invention (as above-mentioned chimeric 2B6 or 3H7) inject altogether.Preferably, the antibody amount of application is 4 μ g/g mouse body weight (mbw).Administration of antibodies 2 μ g weekly after using for the first time continue 4-6 week.Per two weeks replenish once the human son of imitating and answer cell.One group of mouse will not be given therapeutic antibodies, but be applied the chimeric 4D5 that contains N297A sudden change and IgG 1, respectively as the isotype control antibodies of antitumor and anti-Fc γ RIIB antibody.Mouse can be divided into 4 groups, monitors weekly three times.

Following table 3 is according to the present invention tumour to be removed the exemplary arrangement of studying.As shown in table 3,6 groups of mouse (8 every group) are used for detecting the effect that antibody of the present invention is removed in tumour, have wherein used the combination of target cell and effector cell, and have used two kinds of different antibody concentration combinations.In the A group, only inject tumour cell; In the B group, injection tumour cell and monocyte; In the C group, injection tumour cell, monocyte, anti-tumour antibody (ch4D5); In the D group, injection tumour cell, monocyte, anti-tumour antibody and anti-Fc γ RII antibody; In the E group, injection tumour cell, monocyte and anti-Fc γ RIIB antibody; In the F group, injection tumour cell, monocyte, Ch4D5 (N297Q) and IgG 1.One of ordinary skill in the art will be understood that the different antibodies concentration of different antibodies combination can be detected in above-mentioned tumor model.Preferably, use breast cancer cell line (as SKBR3) carries out the parallel study in the above-mentioned experiment.

Table 3: exemplary experiment scheme in the mouse

8 mouse/groups	The 0th day subcutaneous vaccination tumour cell	The abdominal cavity gave monocyte in the 1st day	The 1st day intraperitoneal gives the ch4D5 of 4 μ g/gm mouse body weight	The 1st day intraperitoneal gives the ch4D5N297Q of 4 μ g/gm mouse body weight	Intraperitoneal gave 4 μ g/gm mouse body weight ch2B6N-297Q in the 1st day	The 1st day intraperitoneal gives the human IgG1 of 4 μ g/gm mouse body weight
8 mouse/groups	The 0th day subcutaneous vaccination tumour cell	The abdominal cavity gave monocyte in the 1st day					A	+	-	-	-	-	-
B	+	+	-	-	-	-	A	+	-	-	-	-	-
B	+	+	-	-	-	-	C	+	+	+	-	-	-
D	+	+	+	-	+	-	C	+	+	+	-	-	-
D	+	+	+	-	+	-	E	+	+	-	-	+	-
F	+	+	-	+	-	+	E	+	+	-	-	+	-

Use the known method of one of ordinary skill in the art, the terminal point of determining the heterograft tumor model according to the histological chemistry and the histopathological examination of tumour size, mouse body weight, survival time and cancer.Every group of mouse is all with evaluated in the table 3.Preferably, check mouse weekly 3 times.The standard of tumor growth can be an abdominal distension, occurs tangible lump in the abdominal cavity.Preferably, calculate the implantation back tumor weight of every day.Above-mentioned standard and other the above-mentioned standard of organizing mouse of D being organized mouse compare, to determine the effect of antibody of the present invention in strengthening the tumour removing.Preferably, the animal of antibody treatment will be than control group how monitored 2 months.

In selectable embodiment, the human Fc gamma RIIB that expresses human Fc gamma RIIB in mouse effector cell knocks in the activity in vivo that mouse can be used to determine antibody of the present invention, and does not need suitably transfection effector cell.Can express the initial mouse (founder mice) of human Fc gamma RIIB by the Fc γ RIIB locus preparation of human Fc gamma RIIB being knocked in mouse.Then, this initial mouse subsequently with backcross to blank background and express human Fc gamma RIIB acceptor.The mouse effector cell that obtains will be expressed endogenous excitability Fc γ RI and Fc γ RIIIA and inhibition human Fc gamma RIIB acceptor.

The activity in vivo of humanized antibody of the present invention can have human primary tumo(u)r derived cell, is further detected in the heterograft mouse model as former ovarian cancer of the mankind and mammary cancer derived cell.Utilize the known method of one of ordinary skill in the art, can detect the expression of Her2/neu in cancer patients's ascites and the serothorax.Sample from ovarian cancer patients can be processed as follows: 20 fens kinds of centrifugal ascites under 40 ℃, 6370g, splitting erythrocyte and use the PBS washed cell.In case detect the expression of Her2/neu in tumour cell, two samples selecting moderate to express and highly express are set up the heterograft tumor model as subcutaneous plantation.Then, isolating tumour cell is come amplifying cells to injection in the mouse peritoneal.About 10 mouse can carry out intraperitoneal injection, and the ascites of every mouse is further changed over to two mouse and obtains the ascites of totally 20 mouse, and this ascites can be used in one group of 80 mouse of injection.The serothorax sample can use with the similar method of ascites and handle.Her2/neu from the serothorax sample ⁺Tumour cell can be injected into mouse and go up right and last left side breast pad.

In some embodiments, if the oncocyte per-cent in ascites or the serothorax sample is lower than other cellular component, this oncocyte in vivo then can increase.In other embodiments, can use the magnetic bead of foregoing CC49 antibody (anti-TAG-72) bag quilt to come the purifying tumour cell, referring to people such as Barker, 2001, Gynecol.Oncol.82:57,63, quoted in full and included in the present invention.In brief, the magnetic bead of CC49 antibody sandwich can be used for separating ovarian tumor cell, and this cell can separate with magnetic bead after 37 ℃ of incubated overnight.In some embodiments,, then use passive get rid of (negativedepletion) to come this tumour cell of enrichment, for example use Stem Cell Technologies, the antibody cocktail that Inc.Canada provides if tumour cell lacks TAG-72 antigen.

In other embodiments, can use other tumor markers outside the Her2/neu to separate tumour cell and non-tumor cell in ascites and the serothorax sample.For serothorax or mammary tissue, reported that in the recent period CD44 (a kind of adhesion molecule), B38.1 (a kind of mammary gland/ovarian cancer specificity marker thing), CD24 (a kind of adhesion molecule) can be used as mark, referring to Al Hajj, Deng the people, 2003, Proc.Natl.Acad.Sci.USA 100:3983,8; The document is quoted in full includes the present invention in.Can be increased to mouse by subcutaneous injection after tumour cell is purified.

Preferably, patient's ascites and serothorax being carried out immunohistochemistry and histological chemistry tests and analyzes neoplastic constitutional features.These methods are that one of ordinary skill in the art are known and be included in the scope of the invention.Can comprise by monitored mark, for example: cytokeratin (identification ovary true tumor and from the mesothelial cell of inflammatory cell and mesenchymal cell); Calretinin (compartment chrotoplast from the positive oncocyte of Her2neu); With CD45 (from other cell mass of sample, separating inflammatory cell).Other can comprise CD3 (T cell), CD20 (B cell), CD56 (NK cell) and CD14 (monocyte) by monitored mark.One of ordinary skill in the art will be understood that, any tumour cell that above-mentioned immunohistochemistry and histochemical method can be used in the inventive method by similar application.After the subcutaneous injection of tumor cells, the clinical and anatomical variations of monitoring mouse.As needs, can perform an autopsy on sb to determine the relation of total tumour between taking place to locate to mouse with the specificity organ.

In a specific embodiments, utilize the ascites of tumor cell line such as IGROV-I, OVCAR-8, SK-B and OVCAR-3 cell and human ovarian carcinoma and patient with breast cancer's serothorax to set up tumour.Preferably, contain in the ascites effector cell and tested antibodies at the tumour target.The human monocyte will be used as the effector cell transfer.

5.3 prevention and methods of treatment

The present invention includes methods of treatment based on antibody, this method comprises one or more humanized antibodies of the present invention is applied to animal, is preferably Mammals, most preferably is the mankind, be used to prevent, treat or improve and unusual Fc γ RIIB level or relevant disease, illness or the relevant symptom of infection of activity, and/or the logical effect that becomes the immunologic function relevant with Fc γ RIIB activity or strengthen the cytotoxic activity or the raising immune composition of second therapeutic antibodies.In some embodiments, methods of treatment and one or more therapies (for example, including but not limited to: chemotherapy, radiotherapy, hormonotherapy and/or biotherapy/immunotherapy) of using one or more antibody of the present invention united use.

Have been found that Fc γ RIIB (CD32B) expresses: fat in following types of organization, the b cell, bone, brain, cartilage, colon, incretory gland, eye, the embryo, gi tract, apparatus urogenitalis, sexual cell, H﹠N, kidney, lung, lymphoglandula, lymphatic reticular endothelial cell, mammary gland, muscle, neural, ovary, pancreas, pancreas islet, hypophysis, placenta, retina, skin, soft tissue, synovial membrane and uterus (data from CancerGenome Anatomy Project of the National Cancer Institute).Therefore, the enough activity of coming Fc γ RIIB in excitement or the above-mentioned any tissue of antagonism of humanization antibody capable of the present invention.For example, Fc γ RIIB placenta express and IgG be transitted to fetus and remove in the immunocomplex process and work (people such as Lyden, 2001, J.Immunol.166:3882-3889).In specific embodiments of the present invention, humanization Fc γ RIIB antibody can be as aborticide.

Prevention of the present invention and therapeutic compound be including, but not limited to the protein molecule, includes but not limited to peptide, polypeptide, protein (comprising protein, antibody of posttranslational modification etc.); Small molecules (less than 1000 dalton), inorganic or organic compound; Nucleic acid molecule includes but not limited to two strands or single stranded DNA, two strands or single stranded RNA and triple helix nucleic acid molecule.Prevention and therapeutic compound can derive from any known organism (including but not limited to: animal, plant, bacterium, fungi, protobiont or virus) or synthetic molecules storehouse.

Humanized antibody can provide by prior art pharmaceutically acceptable composition forms known or disclosed by the invention.As detailed below, humanization antibody capable of the present invention is enough in treatment cancer (particularly strengthening the effectiveness of passive immunotherapy or cancer vaccine), autoimmune disorder, diseases associated with inflammation or allergy (for example, strengthening the effectiveness that is used for the treatment of vaccine hypersensitive).

Humanized antibody of the present invention as the preventing and/or treating property preparation of disease, illness or infection can be applied animal, is preferably Mammals, most preferably is the mankind, prevents, treats or improvement and disease, illness or infect relevant symptom.Antibody of the present invention can be co-administered with one or more other preventing and/or treating property preparation, described other preventing and/or treating property preparation is used to prevent, treat or control companion and unusual Fc γ RIIB level or active relevant disease, illness or infection, and/or by changing the active relevant treatable disease of immunologic function, illness or infection with Fc γ RIIB.In certain embodiments, one or more antibody of the present invention and one or more other therapeutic preparations that is used for the treatment of cancer are applied to animal simultaneously, are preferably the mankind.Term " simultaneously " is not limited to prevent or therapeutic preparation was used in the same time of strictness, and be meant that antibody of the present invention and other antibody are applied acceptor with a definite sequence and the timed interval, so that antibody can provide than the better effect of other administering mode with other preparation acting in conjunction.For example, prevention or therapeutic preparation can be used with any order at one time or at different time points; But if not using at one time, they should be used in the enough close time, so that desired therapeutic or preventive effect are provided.Every kind of therapeutic preparation can be used with any suitable formulation and approach respectively.

In different embodiments, the administration interval between prevention or the therapeutic preparation is: be less than 1 hour, be about 1 hour, about 3 hours of about 2 hours of about 1-, about 2-, about 5 hours of about 4 hours of about 3-, about 4-, about 7 hours of about 6 hours of about 5-, about 6-, about 9 hours of about 8 hours of about 7-, about 8-, about 11 hours of about 10 hours of about 9-, about 10-, about 12 hours of about 11-, be no more than 24 hours or be no more than 48 hours.In the preferred embodiment, two or more components are applied in once visiting the patient.

Dosage of the present invention and frequency are comprised in term treatment effectively and in the prevention effectively.According to each patient's actual conditions and according to seriousness and type, route of administration and patient's age, body weight, reaction and the medical history of the concrete treatment that is given or preventative reagent, cancer, this frequency can be different with dosage.One of ordinary skill in the art can select suitable treatment plan after considering the dosage of these factors and reference literature report and Physician ' s DeskReference (56th ed., 2002) recommendation.

Humanized antibody of the present invention can also be advantageously and other mono-clonal or chimeric antibody, Fc fusion rotein combined utilization, perhaps, strengthen Fc γ RIIB (for example strengthening quantity or activity) and enhancing immunity reaction with the interactional effector cell of antibody with lymphokine, cytokine or hemopoieticgrowth factor (as IL-2, IL-3, IL-4, IL-7, IL-10 and TGF-β) combined utilization.In certain embodiments, cytokine and anti-Fc γ RIIB antibody yoke close.

Humanized antibody of the present invention can also advantageously be used for the treatment of the medication combined use of disease, illness or infection with one or more, described medicine such as carcinostatic agent, anti-inflammatory agent or antiviral agent are as hereinafter describing in detail in 5.4.6 joint and the 5.4.5 joint.

5.3.1 cancer

Humanized antibody of the present invention can use separately or unite use with other therapeutic antibodies well known in the prior art, prevents, suppresses or reduce the growth or the tumor cell transfer of primary tumo(u)r.In one embodiment, humanization antibody of the present invention can with the antibody combined application used in the cancer immunotherapy.The present invention includes antibody of the present invention and another kind of therapeutic antibodies are united use, effector function such as ADCC, the CDC by strengthening described therapeutic antibodies, engulf, conditioning waits the effect that strengthens this para-immunity treatment.Though be not subjected to the restriction of any particular mechanism of action, antibody blocking Fc γ RIIB of the present invention, the Fc γ RIIB on monocyte and the scavenger cell particularly, thus the clinical therapeutic efficacy of tumor specific antibody strengthened, for example strengthened removing by the tumour of excitability Fc γ R mediation.

Accordingly, by combining with cancer antigen-specific with other and having the Cytotoxic antibody combined antibody of the present invention of using, the invention provides prevention or treat the cancer treatment method that is feature with this cancer antigen.Humanized antibody of the present invention can be used for prevention or treatment cancer, particularly the cytotoxic activity that has a Cytotoxic cancer antigen-specific therapeutic antibodies by raising strengthens the tumor cytotoxicity effect of antibody of the present invention, and/or strengthens the ADCC activity or the CDC activity of described therapeutic antibodies.In the bright specific embodiments of this law, humanized antibody of the present invention is with the administration of Fc fusion rotein.In a specific embodiments, humanized antibody of the present invention is united when using separately or with the cytotoxicity therapeutic antibodies, with respect to the primary tumo(u)r growth or the transfer that do not give antibody of the present invention, suppress or reduced the growth of primary tumo(u)r or the transfer of cancer cells, suppress or the ratio that reduces as follows: at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20% or at least 10%.In preferred embodiments, antibody of the present invention and cytotoxicity therapeutic antibodies Combined Preparation, with respect to the growth of the primary tumor that does not give this therapeutic antibodies or the transfer of cancer cells, suppress or reduced the growth of primary tumo(u)r or the transfer at least 99% of cancer cells, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, the growth of at least 20% or at least 10% primary tumor or the transfer of cancer cells.

From normally being converted to malignant state is a multistep process that relates to heredity and outside change.In fact, many changes occur in the cell regulating cycle that promotes the deterioration process, make tumour cell can escape the differentiation of end eventually of environment in the normal regulating tissue and the order of dormancy.The infiltration of cancer cells is relevant with some gene with transfer ability, as CSF-I (colony-stimulating factor 1 or macrophage colony stimulating factor).Though be not subjected to the restriction of particular mechanism of action, CSF-I can mediate tumour progression and transfer by assembling scavenger cell in tumor site.Think now, scavenger cell is at the mediation tumour progression with in shifting, by secretion angiogenesis factor (as thymidine phosphorylase, blood vessel endothelium deutero-somatomedin), somatomedin the Urogastron of tumour cell (as can act on) as paracrine factor, and play the nutrition tumour cell and promoted the transfer of tumour cell and invaded blood vessel (referring to people such as Lin, 2001, J.Exp.Med.193 (6): 727-739; People such as Lin, 2002, Journal of Mammary Gland Biology and Neoplasam 7 (2): 147-162; People such as Scholl, 1993, Molecular Carcinogenesis, 7:207-11; People such as Clynes, 2000, Nature Medicine, 6 (4): 443-446; People such as Fidler, 1985, Cancer Research, 45:4714-26).

The present invention includes and use humanized antibody of the present invention to block macrophage-mediated tumour progression and transfer.Antibody of the present invention is used in particular for taking place the treatment that scavenger cell is invaded the solid tumor of profit.By reducing or removing the scavenger cell number that is gathered in tumor locus, antagonistic antibodies of the present invention is used in particular for control (as reducing or removing) tumour cell and shifts.In some embodiments, using humanized antibody of the present invention to control tumour cell separately shifts.Although be not intended to be subjected to the restriction of particular mechanism of action, antagonistic antibodies of the present invention is using separately that the back combine with inhibition Fc γ RIIB on the scavenger cell and effective minimizing scavenger cell number and limit tumour cell and make progress.Because Fc γ RIIB preferred expression comprises the neoplasm invasiveness scavenger cell on activatory monocyte and scavenger cell, antagonistic antibodies of the present invention reduces or the preferred scavenger cell that is positioned at tumor locus of removing.In some embodiments, it is the cancer of feature that humanized antibody of the present invention is used for the treatment of with the CSF-1 overexpression, includes but not limited to: mammary cancer, uterus carcinoma and ovarian cancer.

The present invention further comprises effective minimizing or removes the humanized antibody of other immunocyte (as dendritic cells and B cell) except the scavenger cell of expressing Fc γ RIIB.Utilize antibody of the present invention effectively to reduce or to remove immunocyte, the number that is reduced is 50%, 60%, 70%, 80%, is preferably 90%, most preferably is 99%.Therefore, humanized antibody of the present invention uses separately or unites use with second antibody and strengthened result of treatment, and described second antibody is for example therapeutic antibodies, as anti-tumour antibody, antiviral antibody and antimicrobial antibody.In some embodiments, this therapeutic antibodies has specificity to cancer cells or inflammatory cell.In other embodiments, this second antibody is in conjunction with normal cell.Although be not intended to be subjected to the restriction of particular mechanism of action, when antibody of the present invention was used to separately reduce the immunocyte of Fc γ RIIB expression, cell group was heavily divided, and remaining cell has excitability Fc acceptor, thus the inhibition that alleviates Fc γ RIIB.When uniting use,, strengthen the effectiveness of this second antibody by strengthening the antibody mediated effect subfunction of Fc mediation with second antibody (as therapeutic antibodies).

Cancer that can be by the inventive method and combination treatment or prevention and relative disease are including, but not limited to leukemia, include but not limited to acute leukemia, acute lymphoblastic leukemia, acute myelocytic leukemia such as myeloblast, promyelocyte, myelomonocyte, monocyte, erythroleukemia and myelodysplastic syndrome, chronic leukemia includes but not limited to chronic myelocytic (granulocyte) leukemia, lymphocytic leukemia, hairy cell; Polycythemia vera; Lymphoma, for example but be not limited to: Hokdkin disease, Fei Hejiejinshi disease; Multiple myeloma, for example but be not limited to: SMM, non-secretory myelomatosis, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom ' s macroglobulinemia; The mono-clonal gamma-globulin that meaning is uncertain; Optimum mono-clonal gamma-globulin; Heavy chain disease; Bone and reticular tissue sarcoma, for example but be not limited to: osseous tissue sarcoma (bone sarcoma), osteosarcoma (osteosarcoma), chondrosarcoma, Ewing ' s sarcoma, pernicious giant cells tumour, the fibrosarcoma of bone, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma, fibrosarcoma, Kaposi ' s sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdosarcoma, synovial sarcoma; Cerebral tumor includes but not limited to neurospongioma, astrocytoma, brain stem neurospongioma, ependymoma, oligodendroglioma, non-neurospongioma, acoustic nerve neurilemmoma, craniopharyngioma, medulloblastoma, durosarcoma, pinealoma, pineocytoma, former brain lymphoma; Mammary cancer includes but not limited to gland cancer, leaflet (minicell) cancer, intraductal carcinoma, marrow mammary cancer, Saliva Orthana mammary cancer, tubulose mammary cancer, corpora mammillaria mammary cancer, Paget ' s disease and inflammatory breast cancer; Adrenal carcinoma includes but not limited to pheochromocytoma and adrenocortical carcinoma; Thyroid carcinoma includes but not limited to corpora mammillaria or folliculus thyroid carcinoma, marrow thyroid carcinoma and a change thyroid carcinoma; Carcinoma of the pancreas includes but not limited to insulinoma, gastrinoma, glucagonoma of pancreas, VIPoma, Somat secreting tumor and carcinoid tumor or islet cell tumor; The pituitary body cancer includes but not limited to Cushing ' s disease, prolactin secreting tumor, acromegaly and diabetes insipidus; Ocular tumor includes but not limited to ophthalmomelanoma (as iris melanoma, choroidal melanoma and ciliary body melanoma) and retinoblastoma; Carcinoma of vagina includes but not limited to Squamous Cell Carcinoma, gland cancer and melanoma; Carcinoma vulvae includes but not limited to Squamous Cell Carcinoma, melanoma, gland cancer, rodent cancer, sarcoma and Paget ' s disease; Cervical cancer includes but not limited to Squamous Cell Carcinoma, adenocarcinoid; Uterus carcinoma includes but not limited to carcinoma of endometrium and sarcoma of uterus; Ovarian cancer includes but not limited to epithelial ovarian cancer, borderline tumor, sexual cell and mesenchymoma; The esophageal carcinoma includes but not limited to Squamous Cell Carcinoma, gland cancer, adenoid cystic carcinoma, mucoepidermoid carcinoma and oat cell (minicell) cancer; Cancer of the stomach includes but not limited to gland cancer, fungate (polypoid), ulcer, surface diffusion, disperse, malignant lymphoma, liposarcoma, fibrosarcoma and sarcocarcinoma; Colorectal carcinoma; The rectum cancer; Liver cancer includes but not limited to hepatocellular carcinoma and liver poison cell cancer; Carcinoma of gallbladder includes but not limited to gland cancer; Cholangiocarcinoma includes but not limited to corpora mammillaria, knot shape and diffuse type; Lung cancer includes but not limited to nonsmall-cell lung cancer, Squamous Cell Carcinoma (epidermoid carcinoma), gland cancer, large cell carcinoma and small cell lung cancer; Carcinoma of testis, include but not limited to (representational) gonioma, seminoma of testis, a change, classical, spermatogonium, nonseminoma, embryonal carcinoma cell, teratoma, choriocarcinoma (yolk sac tumor); Prostate cancer includes but not limited to gland cancer, leiomyosarcoma and rhabdosarcoma; The penal cancer; Oral carcinoma includes but not limited to Squamous Cell Carcinoma; Rodent cancer; Salivary-gland carcinoma includes but not limited to gland cancer, mucoepidermoid carcinoma and adenoid cystic carcinoma; The pharynx cancer includes but not limited to Squamous Cell Carcinoma and wart; Skin carcinoma includes but not limited to rodent cancer, Squamous Cell Carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, freckle malignant melanoma, acra melanoma; Kidney includes but not limited to renal cell carcinoma, gland cancer, hypernephroma, fibrosarcoma, transitional cell carcinoma (renal plevis and/or uterus); Wilms ' tumour; Bladder cancer includes but not limited to transitional cell carcinoma, Squamous Cell Carcinoma, gland cancer, sarcocarcinoma.In addition, cancer comprises myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendothelial sarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial cancer, cystadenocarcinoma, bronchogenic carcinoma, syringocarcinoma, sebaceous carcinoma, papillary carcinoma and papillary carcinoma are (about the summary of these diseases, referring to people such as Fishman, 1985, Medicine, 2d Ed., J.B.Lippincott Co., people such as Philadelphia and Murphy, 1997, Informed Decisions:The Complete Book of Cancer Diagnosis, Treatment, andRecovery, Viking Penguin, Penguin Books U.S.A., Inc., United States of America).

The inventive method and composition can also be used for the treatment of or prevent multiple various cancers or other paraplasm disease, include, but is not limited to: comprise the cancer knurl that occurs in following organ: bladder, mammary gland, colon, kidney, liver, lung, ovary, pancreas, stomach, uterine neck, Tiroidina and skin; Squamous Cell Carcinoma; The lymphatic system hematopoietic tumor comprises leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, B cell lymphoma, t cell lymphoma, Berketts lymphoma; The medullary system hematopoietic tumor comprises acute and chronic myelocytic leukemia and promyelocytic leukemia; The tumour in mesenchyme source comprises fibrosarcoma and rhabdosarcoma; Other tumour comprises melanoma, spermocytoma, teratocarcinoma, neuroblastoma and neurospongioma; The tumour of maincenter and peripheral nerve system comprises astrocytoma, neuroblastoma, neurospongioma and schwannoma; The tumour in mesenchymal cell source comprises fibrosarcoma, rhabdosarcoma and osteosarcoma; Other tumour comprises melanoma, painted xeroderma (xenodermapegmentosum), keratoacanthoma (keratoactanthoma), seminoma of testis, thyroid follcular carcinoma and teratocarcinoma.Also should consider, also can treat by the inventive method and composition by the unusual cancer that causes of apoptosis.This class cancer includes but not limited to: follicular lymphoma; The cancer of p53 sudden change; The hormone-dependent tumor of breast tumor, prostate gland and ovary; Precancerous lesion is as familial adenomatous polyposis; And DMPS.In specific embodiments, the malignant tumour or the abnormality proliferation for the treatment of in ovary, bladder, mammary gland, colon, lung, skin, prostate gland or the uterus by the inventive method and composition sexually revise (as metaplasias and dysplasia) or abnormality proliferation disease.In other specific embodiment, method and composition of the present invention can be used for treating live prevention sarcoma, melanoma or leukemia.

Can by co-administered antibody of the present invention with in conjunction with cancer antigen and have the cancer that Cytotoxic antibody is treated or prevention is relevant with cancer antigen.In one embodiment, antibody of the present invention has strengthened the cytotoxicity that antibody mediated to the particular cancer antigen-specific.For example (but not being restriction) can treat with composition or prevention and the relevant cancer of following cancer antigen by the inventive method: KS 1/4 pancarcinoma antigen (Perez and Walker, 1990, J.Immunol.142:32-37; Bumal, 1988, Hybridoma7 (4): 407-415); Ovarian cancer antigen (CA125) (people such as Yu, 1991, Cancer Res.51 (2): 48-475); Prostatic acid phosphatase (people such as Tailor, 1990, Nucl.Acids Res.18 (1): 4928); Prostate specific antigen (Henttu and Vihko, 1989, Biochem.Biophys.Res.Comm.10 (2): 903-910; People such as Israeli, 1993, Cancer Res.53:227-230); Melanoma associated antigen p97 (people such as Estin, 1989, J.Natl.Cancer Instit.81 (6): 445-44); Melanoma antigen gp75 (people such as Vijayasardahl, 1990, J Exp.Med.171 (4): 1375-1380); High molecular melanoma antigen (HMW-MAA) (people such as Natali, 1987, Cancer59:55-3; People such as Mittelman, 1990, J.Clin.Invest 86:2136-2144); Prostate specific membrane antigen; Carcinomebryonic antigen (CEA) (people such as Foon, 1994, Proc.Am.Soc.Clin.Oncol.13:294); Multiform epithelium mucin antigen; HMFG's antigen; The colorectum tumor associated antigen, CEA, TAG-72 (people such as Yokata for example, 1992, Cancer Res.52:3402-3408), CO17-1A (people such as Ragnhammar, 1993, Int.J.Cancer 53:751-758), GICA 19-9 (people such as Herlyn, 1982, J.Clin.Immunol.2:135), CTA-I and LEA; Burkitt ' s lymphoma antigen-38.13, CD19 (people such as Ghetie, 1994, Blood 83:1329-1336); Human B lymphoma antigen-CD20 (people such as Reff, 1994, Blood83:435-445), CD33 (people such as Sgouros, 1993, J.Nucl.Med.34:422-430); Melanoma specific antigens such as Sphingolipids,sialo GD2 (people such as Saleh, 1993, J.Immunol., 151,3390-3398), Ganglioside, GD3 (people such as Shitara, 1993, Cancer Immunol.Immunother.36:373-380), Ganglioside GM2 (people such as Livingston, 1994, J.Clin.Oncol.12:1036-1044), Ganglioside GM3 (people such as Roon, 1993, Cancer Res.53:5244-5250); Tumour-specific transplantation type cell-surface antigens (TSTA) as the tumour antigen of virus induction, comprises T antigen of DNA tumour virus and the envelope antigen of RNA tumour virus; Carcinomebryonic antigen such as alpha-fetoprotein, as the CEA of Crohn disease, tumor of bladder carcinomebryonic antigen (people such as Hellstrom, 1985, Cancer.Res.45:2210-2188); Differentiation antigen is as Human Lung Cancer antigen L6, L20 (people such as Hellstrom, 1986, Cancer Res.46:3917-3923); Fibrosarcoma antigen; Human non-leukemia T cell antigen Gp37 (people such as Bhattacharya-Chatterjee, 1988, J.oflmmun.141:1398-1403); Neural glycoprotein; Sphingolipid; Breast cancer antigen is as EGFR (EGF-R ELISA), HER2 antigen (p185HER2); Polymorphic epidermis Saliva Orthana (PEM) (people such as Hilkens, 1992, Trends in Bio.Chem.Sci.17:359); Pernicious human lymphocyte antigen APO-1 (people such as Bernhard, 1989, Science 245:301-304); Differentiation antigen (Feizi, 1985, Nature314:53-57), as: the I type antigen of finding in fetus red blood corpuscle and the primary endoderm, the I that finds in the adenocarcinoma of stomach (Ma), the M18 and the M39 that find in the breast epithelium tissue, the SSEA-I that finds in the medullary cell, the VEP8 that finds in the colorectal carcinoma, VEP9, MyI, VIM-D5 and D ₁56-22, the TRA-1-85 that finds in the adenocarcinoma of colon (blood group H), C14, the F3 that finds in the adenocarcinoma of lung, the AH6 that finds in the cancer of the stomach, the Y haptens, the Le that find in the embryo cells ^y, the TL5 that finds in the A431 cell (blood group A), EGF acceptor, the E that finds in the prostate cancer ₁System (blood group B), the FC 10.2 that finds in the embryo cells, CO-514 (the blood group Le that finds in the gland cancer ^a), the NS-10 that finds in the gland cancer, CO-43 (the blood group Le that finds in the adenocarcinoma of colon ^b), G49, EGF acceptor (blood group ALe ^b/ Le ^y), find in the colorectal carcinoma 19.9, the cancer of the stomach Saliva Orthana, the T5A7 that in medullary cell, finds, the R24 that finds in the melanoma, find in the embryo cells 4.2, G _D3, Dl.1, OFA-I, G _M2, OFA-2, G _D2, the Ml:22:25:8 that in embryo cells, finds, and the SSEA-3, the SSEA-4 that find in the 4-8 cell stage fetus.In the another one embodiment, described antigen is the TXi Baoshouti derived peptide (referring to Edelson, 1998, The CancerJournal 4:62) from cutaneous T cell lymphoma

Humanized antibody of the present invention can be united to make and is used for strengthening result of treatment with any therapeutic anticancrin well known in the prior art.For example, humanization antibody of the present invention can with listed any antibody combined use in the table 4, these antibody are verified in oncotherapy to have a therapeutic action.By strengthening at least a antibody-mediated effector function of described cancer therapy antibody, humanized antibody of the present invention has strengthened the result of treatment of cancer therapy antibody.In a specific embodiments, rely on cascade reaction by the complement that strengthens described cancer therapy antibody, humanized antibody of the present invention strengthens result of treatment.In the another one embodiment, humanized antibody of the present invention strengthens result of treatment by strengthening to engulfing with opsonization of target tumour cell.In the another one embodiment, antibody of the present invention strengthens result of treatment by the cytotoxicity (" ADCC ") that strengthens the antibody dependent cellular mediation in eliminating target tumour cell process.

Antibody of the present invention can also with unite use based on the product of cytosine(Cyt)-guanine dinucleotides (" CpG "), described product is developed (Coley Pharmaceuticals) and comes out as the activator of heredity and the acquired immune response.For example, the present invention includes CpG 7909, CpG 8916, CpG 8954 (ColeyPharmaceuticals) makes at method and composition of the present invention and is used for treating and/or preventing cancer (referring to people such as Warren, 2002, Semin Oncol., 29 (1 Suppl 2): 93-7; People such as Warren, 2000, ClinLymphoma, 1 (1): 57-61, both of which are incorporated herein by reference, it is introduced in full includes the present invention in).

Humanized antibody of the present invention can be united with the therapeutic antibodies that does not mediate its therapeutic activity by cell killing makes the therapeutic action that is used for strengthening this antibody.In a specific embodiments, the present invention includes antibody of the present invention and unite use with antibody (as anti-Fas antibody) with therapeutic cell death inducing of agonist activity.Anti-Fas antibody is well known in the prior art, comprising: for example Jo2 (people such as Ogasawara, 1993, Nature 364:806) and HFE7 (people such as Ichikawa, 2000, Int.Immunol.12:555).Though be not intended to be subjected to the restriction of particular mechanism of action, Fc γ RIIB participated in promoting in the apoptosis of anti-Fas mediation, referring to: people such as Xu, 2003, Journal of Immunology, 171:562-568.In fact, the cell outskirt of Fc γ RIIB can be used as the linking agent of Fas acceptor, forms functional complex and promotes Fas dependent cell apoptosis.In some embodiments, antibody blocking of the present invention the interaction of anti-Fas antibody and Fc γ RIIB, the active decline of apoptosis that causes the Fas mediation.The active antibody of the present invention that descends of apoptosis that causes Fas mediation with have adverse side effect such as hepatotoxic anti-Fas antibody is united use.In other embodiments, antibody of the present invention has strengthened the interaction of anti-Fas antibody and Fc γ RIIB, causes the apoptosis increased activity of Fas mediation.Antibody of the present invention has the enhanced result of treatment with the antibody combined use with therapeutic cell death inducing of agonist activity.

The antibody of the therapeutic cell death inducing that uses in the inventive method can specificity at any death receptor that is used to regulate apoptotic pathways well known in the prior art, as the TNFR receptor family.

The invention provides the method for the disease (as cancer or autoimmune disorder) of treatment apoptosis mediation signal weakening.In specific embodiments, the present invention includes the method that treatment lacks the apoptotic disease of Fas mediation, described method comprises co-administered antibody of the present invention and anti-Fas antibody.

In some embodiments, agonistic antibody of the present invention is used in particular for treating the tumour in non-hematopoietic cell source, comprises the tumour of melanoma cell.Although be not intended to be subjected to the restriction of particular mechanism of action, the effectiveness of agonistic antibody of the present invention part is owing to the activation of Fc γ RIIB inhibition approach, because the tumour (comprising melanoma cell) in non-hematopoietic cell source is expressed Fc γ RIIB.In fact, nearest experiment shows, in the melanoma cell expression of Fc γ RIIB with the direct interaction (as by combining) of dependence mode in the cytoplasm and anti-tumour antibody with the Fc district of anti-tumour antibody thus regulate tumor growth (people such as Cassard, 2002, Journal of Clinical Investigation, 110 (10): 1549-1557).

In some embodiments, the present invention includes antibody of the present invention combines tumour antigen with immunologic opsonin therapeutic antibodies and unite use, described tumour antigen is not expressed at tumour cell itself, but is expressed in reactivity on every side and comprises on the supportive non-malignant cell of tumour of mesenchyma stroma of tumors.Mesenchyma stroma of tumors comprises the endotheliocyte that forms neovascularity and around matter inoblast between tumor vessels.In specific embodiments, antibody of the present invention combines the antibody combined use of the tumour antigen of endothelial cell surface with immunologic opsonin.In a preferred embodiment, antibody of the present invention and immunologic opsonin are combined into the antibody combined use of the tumour antigen (as fibroblast activation protein (FAP)) on fibrocyte surface.FAP is the homodimer II type glycoprotein of 95KDa, its highly be expressed in many solid tumors between in the matter inoblast, include but not limited to: lung cancer, mammary cancer and colorectal carcinoma are (referring to people such as Scanlan, 1994; Proc.Natl.Acad.USA, 91:5657-61; People such as Park, 1999, J.Biol.Chem., 274:36505-12; People such as Rettig 1988, Proc NatlAcad Sci USA 85:3110-3114; People such as Garin-Chesea, 1990, Proc.Natl.Acad.Sci.USA87:7235-7239).With FAP immunologic opsonin bonded antibody be that prior art is known and be included in the scope of the present invention, referring to people such as Wuest, 2001, Journal of Biotechnology, 159-168; People such as Mersmann, 2001, Int.J.Cancer, 92:240-248; U.S. Patent number 6,455,677; All documents are quoted in full includes the present invention in.

Recently, IgE is proved to be the regulatory factor that can be used as tumor growth, and in fact, it is that (relevant summary is referring to people such as Mills for the natural mechanism that participates in antitumor reaction that the reaction of the quick allergy of IgE target and allergic inflammation has been suggested, 1992, Am.Journal of Epidemiol.122:66-74; People such as Eriksson, 1995, Allergy 50:718-722).In fact, recent research shows, has loaded the tumour cell meeting degrowth of IgE, causes the repulsion of tumour sometimes.According to this research, the tumour cell that has loaded IgE not only has the treatment potentiality, and possesses secular antineoplastic immune, comprise and activate congenital immunity effector mechanism and the cell-mediated the acquired immune response of T, referring to people such as Reali, 2001, Cancer Res.61:5516-22; Quoted in full and included in the present invention.Treatment for cancer and/or the prevention in, antagonist antibody of the present invention can with the co-administered IgE cancers mediated result of treatment that strengthens of IgE.Though be not intended to be subjected to the restriction of particular mechanism of action, antibody of the present invention has strengthened IgE treatment tumor treatment by the blocking-up inhibitory pathway and has renderd a service.With respect to independent use IgE treatment cancer, antagonist antibody of the present invention can pass through: (i) enhancing delays tumor growth; (ii) reduce tumour progression speed; (iii) strengthen repulsion to tumour; Or (iv) strengthen preventative immunity, strengthen the effect of IgE cancers mediated therapy.

Cancer therapy reagent and dosage thereof, route of administration and recommendation usage are that prior art is known and be disclosed in the document, for example referring to Physician ' s Desk Reference (56 editions, 2002, quoted in full and includes this paper in).

5.3.2 B cell malignancies

Agonistic antibody of the present invention can be used for treatment or prevents any B cell malignancies, particularly non_hodgkin lymphoma and chronic lymphocytic leukemia.Other B cell malignancies comprises small lymphocyte lymphoma, Burkitt ' s lymphoma, lymphoma mantle cell, disperse SCC lymphoma, most of follicular lymphoma and some dispersivity large B cell lymphoid tumors (DLBCL).Fc γ RIIB be the unusual target spot that chromosome translocation causes in the malignant lymphoma (particularly B cell non-Hodgkin's) (referring to people such as CallananM.B., 2000 Proc.Natl.Acad.Sci.U.S.A., 97 (1): 309-314).Therefore, antibody of the present invention can be used for treating or preventing the chronic lymphocytic leukemia (summary referring to: Freedman, 1990, Hemtaol.Oncol.Clin.North Am.4:405) of any B clone.Though be not intended to be subjected to the restriction of any mechanism of action, agonistic antibody of the present invention is by suppressing B cell proliferation and/or activation and suppress or having prevented the B cell malignancies.The present invention comprises also that agonistic antibody of the present invention and known other methods of treatment of prior art (as chemotherapy and radiotherapy) unite to make and is used for treating and/or preventing the B cell malignancies.The present invention comprises that also agonistic antibody of the present invention and known other antibody combined the making of prior art are used for treating and/or preventing B cell lymphoma.For example, agonistic antibody of the present invention can with following antibody combined use: anti-C22 or anti-CD19 (people such as Goldenberg is open, U.S. Patent number 6,306,393), anti-CD20 antibodies, anti-CD 33 antibody or anti-CD 52 antibody.

Antibody of the present invention can also be united use with for example following preparation: Oncoscint (target spot: CEA), Verluma (target spot: GP40), Prostascint (target spot: PSMA), CEA-SCAN target spot: CEA), Rituxin (target spot: CD20), Herceptin (target spot: HER-2), Campath (target spot: CD52), Mylotarge (target spot: CD33), LymphoCide (target spot: CD22), Lymphocide Y-90 (target spot: CD22) and Zevalin (target spot: CD20).

5.3.3 autoimmune disorder and inflammatory disease

Agonistic antibody of the present invention can be used for treating or prevention autoimmune disorder or Inflammatory response.The invention provides and be used to prevent, treat or the method for one or more symptoms that control is relevant with patient's autoimmune disorder or inflammatory diseases, comprise antibody of the present invention or its fragment to described patient's administering therapeutic significant quantity.The present invention also provides and has been used to prevent, treat or the method for one or more symptoms that control is relevant with patient's inflammatory diseases, further is included as one or more anti-inflammatory preparations of described patient's administering therapeutic significant quantity.The present invention also provides and has been used to prevent, treat or the method for one or more symptoms that control is relevant with patient's autoimmune disorder, further is included as one or more immunomodulators of described patient's administering therapeutic significant quantity.5.4.5 joint provides the non-limiting of anti-inflammatory agent and immunomodulator to give an example.

Humanized antibody of the present invention can also with the antibody combined use that is used for the treatment of and/or prevents autoimmune disorder or inflammatory diseases well known in the prior art.The limiting examples that is used for the treatment of or prevents the antibody of inflammatory diseases or Fc fusion rotein shown in table 4A, be used for the treatment of or the limiting examples of prevent antibody of autoimmune disorder or Fc fusion rotein as showing shown in the 4B.Humanized antibody of the present invention can strengthen the therapeutic antibodies shown in table 5A and the 5B or the result of treatment of Fc fusion rotein.For example, but unrestricted, antibody of the present invention can strengthen the immune response of the individuality of any antibody shown in acceptance table 5A or the 5B or the treatment of Fc fusion rotein.

Humanized antibody of the present invention can also be united use with following substances, for example but non-being limited to: OrthocloneOKT3, ReoPro, Zenapax, Simulec, Rituximab, Synagis and Remicade.

Humanized antibody of the present invention can also with unite use based on the product of cytosine(Cyt)-bird pyrimidine dinucleotides (" CpG "), this product is developed (ColeyPharmaceuticals) as congenital and promotor the acquired immune response or is being developed.For example, present invention resides in and use CpG 7909, CpG 8916, CpG 8954 (Coley Pharmaceuticals) in the inventive method and the composition, to treat and/or prevent autoimmune disorder or inflammatory diseases (people such as Weeratna, 2001, FEMS Immunol MedMicrobiol., 32 (1): 65-71 is quoted in full and is included in the present invention).

Can include but not limited to by the example of using the autoimmune disorder that antibody of the present invention treats: alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, the autoimmunity Addison's disease, the suprarenal gland autoimmune disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and testitis, AT, Behcet ' s disease, bullous pemphigoid, myocardosis, belly stomatitis dermatitis (celiac sprue dermatitis), confirmed fatigue immunodeficiency disease syndrome (CFIDS), the chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome (allergic granulomatous vasculitis), cicatricial pemphigoid, CREST syndrome, the plain disease of condensation, Crohn disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves ' disease, Guillain-Barre, struma lymphomatosa, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), the IgA DPN, juvenile arthritis, lichen planus, lupus erythematosus, plum Ni Ershi disease, mixed connective tissue disease, multiple sclerosis, I type or immune-mediated diabetes, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, the rheumatic polymyopathy, PM-DM, primary agammaglobulinemia, primary biliary cirrhosis, psoriatic, psoriatic arthritis, raynaud's sign, Reiter ' s syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, sjogren syndrome, the stiff-man syndrome, systemic lupus erythematous, lupus erythematosus, Takayasu arteritis, transience arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis (as dermatitis herpetiformis), vitiligo and Wegener ' s granuloma.The example of inflammatory diseases includes but not limited to: the chronic inflammatory diseases that asthma, encephalitis, inflammatory bowel, chronic obstructive pulmonary disease (COPD), allergic diseases, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated joint disease, sacroiliitis, inflammatory osteolysis and slow virus or infectation of bacteria cause.As described in 3.1 joints, some autoimmune disorders are followed inflammation.Therefore, between autoimmune disorder and inflammatory diseases, exist overlapping.Therefore, some autoimmune disorders also can have the feature of inflammatory diseases.The example of the inflammatory diseases that can prevent, treat or control according to the inventive method includes but not limited to: the chronic inflammatory diseases that asthma, encephalitis, inflammatory bowel, chronic obstructive pulmonary disease (COPD), pathergy illness, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated joint disease, sacroiliitis, inflammatory osteolysis and slow virus or infectation of bacteria cause.

In certain embodiments of the invention, humanized antibody of the present invention can be used for treating and be easier to the autoimmune disorder that takes place in certain sex.For example, the occurred frequently expression with Fc γ RIIB2 of Graves ' disease in the women relevant (people such as Estienne, 2002, FASEB J.16:1087-1092).Therefore, humanized antibody of the present invention can be used for treating, prevent, alleviates or control Graves ' disease.

Humanized antibody of the present invention can also be used to alleviating the particularly mammiferous inflammation of the animal that suffers from inflammatory diseases.In a specific embodiments, with respect to the inflammation of the animal of not using described antibody, the ratio that antibody alleviates the animal inflammation is at least 99%, is at least 95%, is at least 90%, is at least 85%, is at least 80%, is at least 75%, is at least 70%, is at least 60%, is at least 50%, is at least 45%, is at least 40%, is at least 35%, is at least 30%, is at least 25%, is at least 20%, is at least 10%.In the another one embodiment, with respect to the animal inflammation of not using described antibody, uniting of antibody uses the ratio that alleviates the animal inflammation to be at least 99%, to be at least 95%, to be at least 90%, to be at least 85%, to be at least 80%, to be at least 75%, to be at least 70%, to be at least 60%, to be at least 50%, to be at least 45%, to be at least 40%, to be at least 35%, to be at least 30%, to be at least 25%, to be at least 20%, to be at least 10%.

Humanized antibody of the present invention can also be used for preventing the generation of transplant rejection.

The table 4A can with the antibody combined antibody that is applied to inflammatory diseases and autoimmune disorder of the present invention

The antibody title	Target antigen	Product type	Isotype	The manufacturer	Indication
The antibody title	Target antigen	Product type	Isotype	The manufacturer	Indication	5G1.1 5G1.1 5G1.1 5G1.1-SC 5G1.1-SC 5G1.1-SC	Complement (C5) complement (C5) complement (C5) complement (C5) complement (C5) complement (C5)	Humanization humanization humanization humanization humanization humanization	IgG IgG IgG ScFv ScFv ScFv	Alexion Pharm Inc Alexion Pharm Inc Alexion Pharm Inc Alexion Pharm Inc Alexion Pharm Inc Alexion Pharm Inc	Rheumatoid arthritis SLE ephritis cardiopulmonary bypass myocardial infarction angiopoiesis

The anti-LFA1 Antova of the anti-CD18 of the anti-CD11a of ABX-CBL ABX-CBL ABX-IL8 Antegren Antova BTI-322 CDP571 CDP571 CDP850 Corsevin M D2E7 Hu23F2G Hu23F2G IC14 ICM3 IDEC-114 IDEC-131 IDEC-131 IDEC-151 IDEC-152 Infliximab Infliximab LDP-01 LDP-01

CBL CD147 IL-8 VLA-4 CD11a CD18 CD18 CD40L CD40L CD2 TNF-α TNF-α E-Selectin FactVII TNF-α CDll/18 CDll/18 CD14 ICAM-3 CD80 CD40L CD40L CD4 CD23 TNF-α TNF-α β 2-integrin β 2-integrin

The chimeric humanization humanization of the chimeric people's humanization of people mouse people humanization humanization humanization mouse humanization humanization rat humanization humanization humanization humanization humanization Primatised humanization humanization Primatised Primansed

IgG IgG2 IgG IgG1 Fab′2 Fab′2 IgG IgG IgG IgG4 IgG4 IgG IgG1 IgG1 IgG1 IgG IgG

Abgenix Inc Abgenix Inc Abgenix Inc Athena/Elan Genentech Inc/Xoma Genentech Inc Pasteur-Merieux/ Immunotech Biogen Biogen Medimmune Inc Celltech Celltech Celltech Centocor Abbott ICOS Pharm Inc ICOS Pharm Inc ICOS Pharm Inc ICOS Pharm Inc IDEC Pharm/Mitsubishi IDEC Pharm/Eisai IDEC Pharm/Eisai IDEC Rheumatoid Pharm/GlaxoSmit h Kline IDEC Pharm Centocor Centocor Millennium Inc(LeukoSite Inc.) Millennium

GvHD homograft rejection psoriasis multiple sclerosis psoriasis miocardial infarction homograft rejection homograft rejection homograft rejection GvHD, psoriasis Crohn's disease rheumatoid arthritis psoriasis anticoagulation rheumatoid arthritis multiple sclerosis apoplexy toxic shock psoriasis psoriasis SLE multiple sclerosis rheumatoid arthritis asthma/allergic reaction rheumatoid arthritis Crohn's disease apoplexy homograft rejection

The anti-CD3 OKT3 of LDP-02 MAK-195F MDX-33 MDX-CD4 MEDI-507 MEDI-507 OKT4A OrthoClone OKT4A OrthoClone/ RepPro/ Abciximab rhuMab-E25 SB-240563 SB-240683 SCH55700 Simulect SMART a-CD3 SMART a-CD3 SMART a-CD3 Zenapax

α4β7 TNF-α CD64(FcR) CD4 CD2 CD2 CD4 CD4 CD3 gpllbllla IgE IL5 IL4 IL5 CD25 CD3 CD3 CD3 CD25

The chimeric humanization humanization humanization humanization of humanization humanization humanization humanization that everybody humanization humanization humanization humanization mouse of humanization mouse is chimeric

Fab′2 IgG IgG IgG mIgG2a Fab IgG1 IgG1 IgG IgG1

Inc(LeukoSite Inc.) Millennium Inc(LeukoSite Inc.) Knoll Pharm， BASF Medarex/Centeon Medarex/Eisai/ Genmab Medimmune Inc Medimmune Inc Ortho Biotech Ortho Biotech Ortho Biotech Cenrocor/Lilly Genentech/Novar tis/Tanox Biosystems GlaxoSmithKline GlaxoSmithKline Celltech/Schering Novartis Pharm Protein Design Lab Protein Design Lab Protein Design Lab Protein Design Lab/Hoffman-La Roche

Ulcerative colitis toxic shock LADA hematopoietic disease rheumatoid arthritis psoriasis GvHD homograft rejection autoimmune disease homograft rejection coronary angioplasty complication asthma/allergic reaction asthma/allergic reaction asthma/allergic reaction asthma/allergic reaction homograft rejection autoimmune disease homograft rejection psoriasis homograft rejection

Table 4B is used for the antibody and the Fc fusion rotein of autoimmune disorder

Antibody	Indication	Target antigen
Antibody	Indication	Target antigen	ABX-RB2 ILl-ra sTNF-RI 5c8 (anti-CD-40 aglucon antibody) IDEC 131 IDEC 151 IDEC 152 IDEC 114 MEDI-507 LDP-02 (anti-b7 mAb) SMART anti-IFN-γ antibody Verteportin Thalomid SeICIDs (selective cytokine inhibitory drugs) IMiDs (immunoregulation medicament) MDX-33 MDX-CD4	Rheumatoid arthritis chronic inflammatory disease II phase clinical trial stopped in October, 99, investigation " side effect " systemic lupus erythematosus (sLE) rheumatoid arthritis asthma psoriasis rheumatoid arthritis; Multiple sclerosis; Crohn's disease; The psoriatic inflammatory bowel; Crohn disease; Ulcerative colitis autoimmune disorder rheumatoid arthritis leprosy-approval listing; Crohn ' s disease; The hematologic disease that the autoimmune disorder autoimmune response that rheumatoid arthritis is general causes; The special property sent out thrombopenia purple plague purpura (ITP); Autoimmune hemolytic anemia treatment rheumatoid arthritis and its	At the T cell, B cell and the antigenic antibody of NK cell surface CBL, whole person's antibody-like reorganization anti-inflammatory albumen soluble tumor necrosis factor α-I receptor from transgenic mouse, the anti-CD40 of blocking-up TNF effect CD-40, the anti-CD4 of humanization, the anti-CD23 of primatesization, the anti-CD80 of primatesization, the high degree of specificity supressor of the supressor phosphodiesterase IN (PDE-4) of the anti-IFN-tumor necrosis factor alpha of the a4b7 integrin receptor on the anti-CD2 white corpuscle of primatesization (lymphocyte) (TNF α), increase the cAMP level, activated protein kinase A (PKA), the blocking-up NK-kB factor is transcribed, prevent TNF α genetic transcription, reduce TNF α and generate the thalidomide analog, suppress the mono-clonal of the anti-CD4 acceptor molecule of monoclonal antibody of the anti-FcRI acceptor of TNF α

VX-497 VX-740 VX-745 Enbrel(etanercept) IL-8 5G1.1 ApogenMP4

Its autoimmune disease autoimmune disorder; Multiple sclerosis; Rheumatoid arthritis; Inflammatory bowel; Lupus; The psoriatic rheumatoid arthritis-specific is at inflammation, participate in immunoreactive chemical signal conduction, the outbreak of inflammation and progress rheumatoid arthritis, class sky bleb (dangerous fash), psoriatic, lupus

Antibody inosine inhibitor, single phosphate dehydrogenase (forms new RNA and the required enzyme of DNA, be used to generate the required Nucleotide of lymphocyte breeding) the ICE inhibitor, il-1 beta inhibitor (saccharase, control causes the immunoreactive path of invasive, regulate cytokine) the kinase whose inhibitor of P38MAP, mitogen activated protein kinase target TNF (tumour necrosis factor), the complete human MAB of anti-IL-8 (interleukin 8) (blocking-up IL-8, the blocking-up Inflammatory response) complement C5 inhibitor recombinant antigen, selective destruction disease-related T cell, apoptosis-induced, no longer attack the human body self cell by the T cell that apoptosis is removed, specific apogen target specific T cells

5.3.4 anaphylaxis

The invention provides the method that is used for the treatment of or prevents the hypersensitivity disorders IgE mediation and/or Fc ε RI mediation in patient's body, comprise of the present invention exciting antibody or its fragment to described patient's administering therapeutic significant quantity.Although be not intended to be subjected to the restriction of particular mechanism of action, antibody of the present invention can be used to suppress the mastocyte activation that Fc ε RI inductive causes acute and late phase allergic responses (people 1997 such as Metcalfe D., Physiol.Rev.77:1033).Preferably, and be used for the treatment of in the prior art and/or prevent the ordinary method of the hypersensitivity disorders of IgE mediation to compare, exciting antibody of the present invention has strengthened result of treatment and/or has reduced side effect.The anaphylactoid ordinary method that is used for the treatment of and/or prevents IgE to mediate includes but not limited to: anti-inflammatory agent (as be used for the oral of asthma and suck reflunomide); Antihistaminic (as being used for allergic rhinitis and atopic dermatitis); Cysteinyl leukotrienes (as being used for the treatment of asthma); Anti-IgE antibodies; With concrete immunotherapy or desensitization reaction.

The anaphylactoid example of IgE mediation includes but not limited to: asthma, allergic rhinitis, gi tract anaphylaxis, Eosinophilia, conjunctivitis, atopic dermatitis, urticaria, allergy, glomerulonephritis.

The present invention includes molecule, as immunoglobulin (Ig), it can be formed mixture with Fc ε RI and human Fc gamma RIIB by through engineering approaches, and promptly specificity is in conjunction with Fc ε RI and human Fc gamma RIIB.Preferably, this quasi-molecule has therapeutic action in the disease of IgE and Fc ε RI mediation.Although be not intended to be subjected to the restriction of particular mechanism of action, the treatment of these through engineering approaches molecules is renderd a service part and is suppressed the ability of mastocyte and basophilic leukocyte function owing to them.

In a specific embodiments, with Fc ε RI and human Fc gamma RIIB specificity bonded molecule be the chimeric protein that contains the binding site of Fc ε RI binding site and Fc γ RIIB.This quasi-molecule can carry out through engineering approaches according to the known standard recombinant dna method of one of ordinary skill in the art.In a preferred specific embodiments, the chimeric fusion protein that uses in the inventive method comprises F (ab ') strand of anti-Fc γ RIIB monoclonal antibody of the present invention, merge in this strand and the zone as bridge, and described bridge is connected to huFc ε the C-terminal of F (ab ') strand of described anti-Fc γ RIIB monoclonal antibody.An exemplary chimeric fusion protein that is used for the inventive method comprises: V _L/ C _H(Fc γ RIIB)-hinge-V _H/ C _H(Fc γ RIIB)-joint-C _Hε 2-C _Hε 3-C _Hε 4.The joint that is used for chimeric molecule can be 5,10, be preferably 15 amino acid lengths.Described length of said joint can be different so that combining of the best between described molecule and Fc γ RIIB and the Fc ε RI to be provided.In a specific embodiments, described joint is one and contains 15 amino acid whose joints that sequence set becomes: (Gly ₄Ser) ₃Although be not intended to be subjected to the restriction of particular mechanism of action, it is folding that flexible peptide linker promotes that the pairing of chain and maximum possible reduce again, also makes chimeric molecule can touch two acceptors (being Fc γ RIIB and Fc ε RI) of cell surface and crosslinked with them.Preferably, described chimeric molecule is cloned in the mammalian expression vector (as pCI-neo) with compatible promotor (as cytomegalovirus promoter).Fusion rotein prepared according to the methods of the invention will contain Fc ε RI (CH ε 2CH ε 3) and Fc γ RIIB (VL/CL, the binding site of-hinge-VH/CH).The nucleotide sequence of encoding said fusion protein preferably is transfected in 293 cells, and uses the known ordinary method of prior art to come purifying secreted albumen.

Can use that one of ordinary skill in the art are known to be used to measure Fc γ R bonded usual way and to detect combining of chimeric molecule and Fc ε RI and Fc γ RIIB.Preferably, for example by suppressing degranulation and the cytoactive restraining effect that antigen drives, chimeric molecule of the present invention has treatment and renders a service in the disease of treatment IgE mediation.Before being applied to the mankind, can in transgenic mice, measure the effect of chimeric molecule of the present invention in the mast cell degranulation of the Fc ε RI mediation that blocking-up IgE drives, this transgenic mice is engineered to express human Fc ε R α and human Fc gamma RIIB.

The invention provides the application of bi-specific antibody in the hypersensitivity disorders that treats and/or prevents IgE mediation and/or Fc ε RI mediation.Bi-specific antibody (BsAb) combines with two different epitopes on being present in synantigen not usually.BsAb has the potential clinical application and has been used for the cell and the bacterial pathogen of target virus, virus infection, and to thrombus transmission thrombolytic agent (Cao Y., 1998 Bioconj.Chem 9:635-644; People such as Koelemij, 1999, J Immunother., 22,514-524; People such as Segal, Curt.Opin.Immunol, 11,558-562).The technology that is used to prepare BsIgG and other relevant bispecific molecule is existing (referring to people such as Carter, 2001 J.of Immunol Methods, 248,7-15; People such as Segal, 2001, J.of Immunol Methods, 248,7-15 originally quotes in full and includes the present invention in).The invention provides bi-specific antibody, this antibody contains a F (ab ') of anti-Fc γ RIIB antibody and an a kind of F (ab ') of existing monoclonal anti huIgE antibody, and described bi-specific antibody is assembled two kinds of acceptor Fc γ RIIB and the Fc ε RI on the same cell surface.Can adopt the known and disclosed by the invention any method cost in next life of prior art to be used for the bi-specific antibody of inventive method.In a specific embodiments, the generation of BsAb be by F (ab ') fragment of above-mentioned anti-Fc γ RIIB antibody and anti-hulgE antibody is carried out chemically crosslinked (referring to people such as Glennie, 1995, Tumor Immunobiology, Oxford University press, Oxford, p.225; Quoted in full and included in the present invention).The segmental generation of F (ab ') can be carried out limited proteolysis and use mercapto-ethanol reduction to provide to have the Fab ' fragment of free hinge area sulfydryl (SH) by stomach en-.(SH) the SH group on the fragment can be by excessive adjacent phenylenedimaleimide (O-PDM) alkylation to provide free maleimide base group (mal) for Fab '.Fab ' (mal) and Fab ' (SH) can suitable proportion generate the heterodimer structure in conjunction with (being preferably 1: 1).BsAb can carry out purifying with molecular exclusion chromatography by the known method of one of ordinary skill in the art, and characterizes with HPLC.

Particularly, the present invention includes and contain first heavy chain-light chain the bi-specific antibody right with second heavy chain-light chain, described first heavy chain-light chain pair and Fc γ RIIB bonded avidity are greater than it and Fc γ RIIA bonded avidity, described second heavy chain-light chain is in conjunction with the IgE acceptor, and condition is that described first heavy chain-light chain is at first combining with Fc γ RIIB.Bi-specific antibody of the present invention can utilize the known standard technique of prior art to carry out through engineering approaches, to guarantee it and the combining prior to it and the combining of IgE acceptor of Fc γ RIIB.One of ordinary skill in the art will appreciate that, need this bi-specific antibody of through engineering approaches, for example make this bi-specific antibody and the Fc γ RIIB bonded avidity avidity greater than described antibody and IgE acceptor.In addition, can pass through this bi-specific antibody of the known engineeringization of prior art, for example by adding joint so that the hinge area length of this antibody increases, make this bi-specific antibody have snappiness, thus on same cell in conjunction with IgE acceptor and Fc γ RIIB.

Humanized antibody of the present invention can also with other therapeutic antibodies well known in the prior art or medication combined use, be used for the treatment of or prevent the hypersensitivity disorders of IgE mediation.For example, antibody of the present invention can with following any medication combined use: azelastine, Astelin, the beclomethasone dipropionate inhalation, Vanceril, beclomethasone dipropionate insufflation/sprays, Vancenase, Budesonide insufflation/sprays of Beconase, the alerlisin of Rhinocort, the chlorphenamine of Zyrtec, pseudo-ephedrine, Deconamine, Sudafed, Sodium Cromoglicate, Nasalcrom, Intal, Opticrom, Desloratadine, Clarinex, fexofenadine and pseudo-ephedrine, Allegra-D, fexofenadine, Allegra removes fluorine fluocinonide nasal spray, Nasalide fluticasone propionate insufflation/sprays, fluticasone propionate oral cavity inhalation, Flovent, hydroxyzine, Vistaril, Ataraxloratadine, pseudo-ephedrine, Zeos (Claritin-D), Loratadine, Claritin, Ultracortene-H (Prednisolone), the prednisone sodium phosphates oral solution, the Medrol prednisone, prednisone (Deltasone), liquid Predsalmeterol, the Triamcinolone Acetonide inhalation of Serevent, Azmacort Triamcinolone Acetonide inhalation/sprays, Nasacort or NasacortAQ.Antibody of the present invention can with unite use based on the product of cytosine(Cyt)-guanine dinucleotides (" CpG "), this CpG has been developed or has been developed as congenital and activator (Coley Pharmaceuticals) the acquired immune response.For example, present invention resides in and use CpG 7909, CpG 8916, CpG8954 (Coley Pharmaceuticals) in the inventive method and the composition, the hypersensitivity disorders that treats and/or prevents IgE mediation is (referring to people such as Weeratna, 2001, FEMS Immunol Med Microbiol., 32 (1): 65-71 is quoted in full and is included in this paper).

The present invention includes the therapeutic antibodies combined utilization of humanized antibody of the present invention and any treatment treatment hypersensitivity disorders well known in the prior art, described therapeutic antibodies such as Xolair ^TM(Omalizumab; Genentech), rhuMAB-E25 (Bio World Today, Nov.10,1998, p.1; Genentech), CGP-51901 (humanization anti-IgE antibodies) or the like.

In addition, the present invention includes the composition combined utilization of known other treatment hypersensitivity disorders of antibody of the present invention and prior art.People's disclosed method and composition (U.S. Patent number 6,426,336 such as Carson for example particularly; U.S. Patent Application Publication No. 2002/0035109 A1 and 2002/0010343, all documents are quoted in full includes the present invention in).

5.3.5 immunomodulator and anti-inflammatory agent

The inventive method provides the method that is used for the treatment of autoimmune disorder and inflammatory diseases, comprises and the co-administered antibody of the present invention of other therapeutic preparation.The example of immunomodulator includes but not limited to: methotrexate, ENBREL, REMICADE ^TM, HUMIRA ^_, leflunomide, endoxan, cyclosporine A, macrolide antibiotics (as FK506 (tacrolimus)), methyl meticortelone (MP), cortin, steroid, mycophenlate mofetil, Wyeth-Ayerst Laboratories (sirolimus), mizoribine, Gusperimus, brequinar, malononitriloamindes (as leflunomide), TXi Baoshouti conditioning agent and cytokine receptor conditioning agent.

Anti-inflammatory agent has been proved to be can successfully treat inflammatory and autoimmune disorder, is the routine and the standard treatments of this class disease at present.The known any anti-inflammatory agent of one of ordinary skill in the art may be used in the method for the present invention.The limiting examples of anti-inflammatory agent comprises: NSAID (non-steroidal anti-inflammatory drug) (NSAID), steroidal anti-inflammatory medicine, β excitomotor, anti-cholinolytic and methyl xanthine.The example of NSAID includes but not limited to: acetylsalicylic acid, Ibuprofen BP/EP, celecoxib (CELEBREX ^TM), diclofenac (VOLTAREN ^TM), R-ETODOLAC (LODINE ^TM), fenoprofen (NALFON ^TM), indomethacin (INDOCIN ^TM), ketorolac (TORADOL ^TM), Taisho) (DAYPRO ^TM), nabumetone (RELAFEN ^TM), sulindac (CLINORIL ^TM), tolmetin (TOLECTIN ^TM), rofecoxib (VIOXX ^TM), Naproxen Base (ALEVE ^TM, NAPROSYN ^TM), Ketoprofen (ACTRON ^TM) and nabumetone (RELAFEN ^TM).This class NSAID plays a role by suppressing cyclooxygenase (as COX-I and/or COX-2).The example of steroidal anti-inflammatory medicine includes but not limited to: glucocorticosteroid, dexamethasone (DECADRON ^TM), cortisone, hydrocortisone, prednisone (DELTASONE ^TM), prednisolone, triamcinolone, sulfasalazine and eicosanoids (as prostaglandin(PG), thromboxane and leukotrienes).

5.3.6 carcinostatic agent and therapeutic antibodies

In a specific embodiments, the inventive method comprises uses one or more angiogenesis inhibitors, such as but not limited to: angiostatin (Profibrinolysin); The Antithrombin III of angiogenesis inhibitor; Angiozyme; ABT-627; Bay 12-9566; Benfluralin (Benefin); RhuMAb-VEGF (Bevacizumab); BMS-275291; Cartilage deutero-inhibitor (CDI); CAI; CD59 complement fragment; CEP-7055; Col 3; Combretastatin A-4; His spit of fland (collagen XVIII fragment) of endothelium; EGFr retarding agent/inhibitor (Iressa_, Tarceva_, Erbitux_ and ABX-EGF); The fibronectin fragment; Gro-β; Halofuginone (Halofuginone); Heparinase; Heparin hexose fragment; HMV833; Human chorionic gonadotropin (hCG); IM-862; Interferon alpha/β/γ; Interferon inducible protein (IP-10); Il-1 2; Kringle 5 (Profibrinolysin fragment); Marimastat bullet (Marimastat); Inhibitors of metalloproteinase (TIMPs); The 2-methoxyestradiol; MMI 270 (CGS27023A); Monoclonal antibody IMC-ICl 1; Neovastat; NM-3; Panzem; PI-88; Placental ribonuclease inhibitor; Type 1 plasminogen activator inhibitor; PF4 (PF4); The prinomastat; Prolactin 16kD fragment; Proliferin-associated protein (PRP); PTK 787/ZK 222594; Retinoid; Solimastat; Squalamine; SS 3304; SU 5416; SU6668; SUl 1248; Urocortisol-S; Tetrathiomolybdate; Thalidomide; Responsive plain-1 (TSP-I) of zymoplasm; TNP-470; Transforming growth factor-beta (TGF-b); Vasculostatin; Vasostatin (calcium net histone fragment); ZD6126; ZD 6474; Farnesyl transferase inhibitor (FTI); And diphosphate.

In a plurality of embodiments of the present invention (comprising pharmaceutical composition of the present invention and formulation and test kit), can include but not limited to: U 42126 with the carcinostatic agent of the antibody combined use of the present invention; Aclarubicin; The hydrochloric acid acodazole; Acronine (acronine); U 73975 (adozelesin); RIL-2 (aldesleukin); Altretamine (altretamine); Duazomycin C (ambomycin); Acetic acid ametantrone (ametantrone acetate); Aminoglutethimide (aminoglutethimide); Amsacrine; Anastrozole; Antramycin; Asparaginase; Asperline; Azacitidine; Azatepa; Azotomycin; Batimastat; Benzodepa (benzodepa); Bicalutamide; Bisantrene hydrochloride; Bisnafide (dimesylate); U 77779; Bleomycin sulfate; Brequinar sodium; Bropirimine; Busulfan; Sanarnycin; Calusterone; Caracemide; Carbetimer (carbetimer); Carboplatin; Carmustine; Carubicin hydrochloride; U 80244; Cedefingol; Chlorambucil; U 12241; Cis-platinum; CldAdo; Crisnatol sideramines (crisnato mesylate); Endoxan; Cytosine arabinoside; Dacarbazine; Dactinomycin; Daunorubicin hydrochloride; Decitabine; U 78938; Dezaguanine; Dezaguanine sideramines (dezaguanine mesylate); Diaziquone; Docetaxel; Dx; Doxorubicin hydrochloride; Droloxifene; K-21060E; Drostanolone; Duazomycin (duazomycin); Edatrexate; Vaniqa; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin hydrochloride; R 55104; Esorubicin hydrochloride; Estramustine (estramustine); Estramustine phosphate sodium; Etanidazole; Etoposide; The phosphoric acid Etoposide; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Ro 2-9757; Fludarabine phosphate; Fluracil; Flurocitabine (flurocitabine); Fosquidone; Phosphotrienin sodium; Gemcitabine; Gemcitabine hydrochloride; Hydroxyurea; Idarubicin hydrochloride; Ifosfamide; Thio ALP; Interleukin I I (comprising recombinant interleukin II or rIL2); Intederon Alpha-2a; Interferon Alpha-2b; Interferon alfa-n1; Alferon N; Interferon beta-Ia; Interferon-gamma-Ib; Iproplatin; Irinotecan hydrochloride; Lanreotide acetate; Letrozole; Leuprorelin acetate; Liarozole hydrochloride; Lometrexol sodium; Lomustine; Losoxantrone hydrochloride; Masoprocol; Maytenin; Mustargen; Magace; Melengestrol acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate sodium; U-197; Meturedepa; Mitindomide; Rice holder jinx (mitocarcin); Mitochromine; Mitogillin (mitogillin); Mitomalcin (mitomalcin); Mitomycin; Mitosper (mitosper); Mitotane; Mitoxantrone hydrochloride; Mycophenolic Acid; R 17934; Nogalamycin; Ormaplatin; Oxisuran (oxisuran); Taxol; Pegaspargase; Peliomycin; Neostigmine bromide; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; The hydrochloric acid piroxantrone; Plicamycin; Plomestane; Porfimer sodium; Porfiromycin; Prednimustine; Procarbazine hydrochloride (procarbazine hydrochloride); Tetracycline (puromycin); Puromycin hydrochloride; Azoles furans rhzomorph (pyrazofurin); Riboprine; Rogletimide; (rogletimide) Safingol; The hydrochloric acid Safingol; Semustine; Simtrazene; Sparfosate sodium; Sparsomycin; Spirogermanium hydrochloride; Spiromustine; Spiral shell platinum; Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Tecogalan sodium; Tegafur (tegafur); Teloxandrone hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; ITG; Tioguanine; Plug is for group; Tiazofurine; Win-59075; Toremifene Citrate; Trestolone acetate; The phosphoric acid triciribine; Trimetrexate (trimetrexate); Glucuronic acid trimetrexate (trimetrexate glucuronate); Triptorelin; Tubulozole hydrochloride; Uramustine (uracilmustard); Uredepa (uredepa); Vapreotide; Visudyne; Vinblastine sulphate (vinblastine sulfate); Vincristine sulphate (vincristine sulfate); Vindesine (vindesine); Ground, sulfuric acid Changchun (vindesinesulfate); The sulfuric acid vinepidine; The sulfuric acid vinglycinate; Vinleurosine sulfate; Vinorelbine tartrate; Vinrosidine sulfate; The sulfuric acid vinzolidine; Vorozole; Zeniplatin; Zinostatin (zinostatin); Zorubicin hydrochloride.Preferred other cancer therapy drug is 5 FU 5 fluorouracil and folinic acid.

The example of the therapeutic antibodies that can use in the methods of the invention includes but not limited to: AVASTIN (rhuMAb-VEGF) (Genentech, South San Francisco, CA), it is the anti-VEGF monoclonal antibody of a kind of humanization, intravenously is used for the chemotherapy based on 5 FU 5 fluorouracil, as treating before colon or colon metastatic carcinoma patient's first-line treatment or the chemotherapy; HERCEPTIN ^_(trastuzumab) (Genentech, South San Francisco, CA), a kind of Humanized anti-HER 2 monoclonal antibody that is used for the treatment of metastatic breast cancer; REOPRO ^_(ReoPro) (Centocor), a kind of antibody that is used to prevent glycoprotein iib/iiia acceptor on the antiplatelet that blood clot forms; ZENAP AX ^_(daclizumab) (RochePharmaceuticals, Switzerland), a kind of Humanized anti-CD 25 monoclonal antibody that is used for prophylaxis of acute kidney allograft rejection; PANOREX ^TM(Edrecolomab), a kind of mouse anti 17-IA cell-surface antigens IgG2a antibody (Glaxo Wellcome/Centocor); BEC2, a kind of mouse anti idiotype (GD3 epitope) IgG antibody (ImClone System); Erbitux ^_(Cetuximab), a kind of inosculating antibody EGFR IgG antibody (ImClone System); VITAXIN ^TM, a kind of humanized anti-alpha V β 3 alphab-integrin antibodies (Applied Molecular Evolution/Medlmmune); Campath 1H/LDP-03, a kind of humanized anti-CD 52 IgG1 antibody (Leukosite); Smart M195, a kind of Humanized CD 3-resisting 3 IgG antibody (Protein Design Lab/Kanebo); RITUXAN ^TM(Mabthera), and a kind of inosculating antibody CD20 IgG1 antibody (IDEC Pharm/Genentech, Roche/Zettyaku); LYMPHOCIDE ^TM(epratuzumab), the anti-CD22 IgG of a kind of humanization antibody (Immunomedics); ICM3, the anti-ICAM3 antibody of a kind of humanization (ICOS Pharm); IDEC-114, a kind of anti-CD80 antibody (IDECPharm/Mitsubishi) of primateization; ZEVALIN ^TM, a kind of radiolabeled mouse anti-CD20 antibodies (IDEC/Schering AG); IDEC-131, a kind of humanization anti-CD40L antibodies (IDEC/Eisai); IDEC-151, a kind of anti-CD 4 antibodies of primateization (IDEC); IDEC-152, a kind of anti-CD23 antibody (IDEC/Seikagaku) of primateization; The anti-CD3 of SMART, a kind of Humanized CD 3-resisting IgG (Protein Design Lab); 5G1.1, a kind of humanization anticomplement factor 5 (C5) antibody (Alexion Pharm); Humira ^_, the anti-TNF-Alpha antibodies of a kind of humanization (Abbott Laboratories); CDP870, the anti-TNF-α of a kind of humanization Fab fragment (Celltech); IDEC-151, a kind of anti-CD4 IgG1 antibody (IDEC Pharm/SmithKline Beecham) of primateization; MDX-CD4, the anti-CD4 IgG of a kind of humanization antibody (Medarex/Eisai/Genmab); CDP571, the anti-TNF-α of a kind of humanization IgG4 antibody (Celltech); LDP-02, a kind of humanized anti-alpha 4 β, 7 antibody (LeukoSite/Genentech); OrthoCloneOKT4A, the anti-CD4 IgG of a kind of humanization antibody (Ortho Biotech); ANTOVA ^TM, a kind of humanization anti-CD 40 L IgG antibody (Biogen); ANTEGREN ^TM, the anti-VLA-4 IgG of a kind of humanization antibody (Elan); And CAT-152, a kind of Humanized anti-TGF-beta 2 antibody (Cambridge Ab Tech).Can be as shown in table 5 with the example of other therapeutic antibodies of the antibody combined use of the present invention.

Table 5: the monoclonal antibody that can treat cancer with the antibody combined use of the present invention

Company	Product	Disease	Target
Company	Product	Disease	Target	Abgenix AltaRex Antisoma Boehringer Ingelheim Centocor/J&J Corixa CRC Technology Crucell Cytoclonal Genentech IDEC ImClone	ABX-EGF OvaRex BravaRex Theragyn (pemtumomabytrrium-90) Therex blvatuzumab Panorex ReoPro ReoPro ReoPro Bexocar monoclonal antibody, idiotype 105AD7 anti-EpCAM MAb Herceptin Herceptin Rituxan Rituxan Avastin^_Avastin ^_AMD Fab E-26(2 ^ndGen.IgE) Zevalin (Rituxan+ Yttrium-90) Cetuximab+innotecan Cetuximab+platinum and radiation	The incidence cancer new diagnosis of colorectal cancer or recurrence of the B cell NHL of the CD20 positive of the low folliculus of the macular degeneration allergic asthma that the minuent of cancer oophoroma metastatic carcinoma oophoroma breast cancer incidence cancer colorectal cancer PTCA acute MI ischemic shock NHL colorectum Theratope Carcinoma metastasis of cancer breast cancer early-stage breast cancer recurrence/refractory or folliculus NHL moderate are relevant with the height NHL metastatic NSCLC metastatic colorectal cancer age and rhinitis, relapsed or stubborn and the NHL refractory of Rituximab tolerance	EGF receptor tumour antigen CA 125 tumour antigen MUCl PEM antigen PEM Antigens CD44 17-1A gpIIIb/IIIa gpIIIb/IIIa gpIIIb/IIIa CD20 gp72 Ep-CAM NA HER-2 HER-2 CD20 CD20 VEGF VEGF CD 18 IgE CD20 EGF acceptor EGF acceptors

ImmonoGen ImmunoMedics Intracel Medarex

Cetuximab+gemcitabine Cetuximab+cis-platinum+5FU or Taxol Cetuximab+carboplatin+taxol Cetuximab+cis-platinum Cetuximab+radiation BEC2+Bacillus Calmette Guerin BEC2+Bacillus Calmette Guerin IMC-ICl 1 nuC242-DMl LymphoCide LymphoCide Y-90 CEA-Cide CEA-Cide Y-90 CEA-Scan (Arcitumomab of Tc-99m-mark) CEA-Scan (Arcitumomab of Tc-99m-mark) CEA-Scan (Arcitumomab of Tc-99m-mark) CEA-Scan (Arcitumomab of Tc-99m-mark) LymphoScan (sulesomab of Tc-99m-mark) LymphoScan (the Tc-99m-mark) AFP-Scan (the Tc-99m-mark) HumaRAD-HN (+Yttrium-90) HumaSPECT MDX-101 (CTLA-4)

EGF EGF EGF ﹠ ( EGF ) EGF GD3 GD3 VEGF 、 nuC242 CD22 CD22 CEA CEA ( CEA ) ( ) CEA ( ) CEA ( CEA ) ( CEA ) ( ) CD22 7 ( AFP ) NA NA CTLA-4

Medlmmune Merck KGaA Millennium NeoRx Peregrine Pharmacia Protein Design Labs Titan Trilex Viventia Biotech Xoma

MDX-210 (her-2 overexpression) MDX-210/MAK Vitaxin MAb 425 IS-IL-2 Campath (alemtuzumab) CD20 antibiosis protein chain mycin (+biotin-Y90) Avidicin (albumin+NRLU 13) Oncolym (+iodine-131) Cotara (+iodine-131) C215 (+staphylococcal enterotoxin) monoclonal antibodies, lung/kidney Nacolomab tafenatox (C242+ staphylococcal enterotoxin) Nuvion SMARTM 195 SMART ID10 CEA Vac TriGem TriAb CEA Vac TriGem TriAb NovoMAb-G2 radiolabeled Monopharm C GHoMAb-H (+gelonin) Rituxan Rituxan ING-1

Degree ﹠ height NHL CD20 suprarenoma Ep-CAM among the sick non_hodgkin lymphoma CD20 metastatic carcinoma NA non_hodgkin lymphoma unresectable pernicious glue dna binding protein dna knurl cancer of pancreas NA lung kidney NA colon of HLA-DR 10 β of the white CD52 blood of the multiple cancer Ep-CAM chronic lymphocytic of prostate cancer HER-2 cancer HER-2 cancer α v β more than 3 kind of cancer EGF acceptor and cancer of pancreas NA T cell malignancies CD3 AML CD33 NHL HLA-DR antigen colorectal cancer CEA metastatic melanoma in late period GD2-gangliosides and ED-SCLC metastatic breast cancer MUC-1 colorectal cancer CEA metastatic melanoma in late period GD2-gangliosides and the straight intestines ﹠ of ED-SCLC metastatic breast cancer MUC-1 non_hodgkin lymphoma NA knot intestines cancer of pancreas SK-1 antigen glioma, melanocyte NA knurl and desmocytoma recurrence/intractable low CD20 or the folliculus NHL

5.3.7 vaccine therapy and prevention

The invention provides and strengthen individual immunoreactive method vaccine composition, this method comprises: use humanized antibody of the present invention or its fragment (itself and Fc γ RIIB specificity bonded avidity are greater than the avidity of it and Fc γ RIIA) and vaccine composition for described individuality, wherein said antibody or its fragment have strengthened individual immune response to described vaccine composition.In a specific embodiments, by strengthening described vaccine institute at antigenic displaying and/or antigen processing, described antibody or its fragment strengthen individual immune response to described vaccine.The known any vaccine composition of prior art can be united use with antibody of the present invention or its fragment.

Though be not intended to be subjected to the restriction of any particular mechanism of action, antibody of the present invention can be blocked at some cell mass and/or some and plant the activation of the Fc γ RIIB that expresses on the dendritic cells, thereby has strengthened the activity of this class dendritic cells in effective seeded process.These enhanced dendritic cells activity can produce prevention or therapeutic vaccine strengthens or better reaction.

In one embodiment, the present invention includes the known any Theratope of humanized antibody of the present invention and prior art and unite use, as Canvaxin ^TM(Cancer Vax, Corporation, melanoma and colorectal carcinoma), Oncophage (HSPPC-96; Antigenics; Metastasis melanin tumor), HER-2/neu Theratope or the like.The Theratope that uses in the inventive method and the composition can be antigen-specific vaccine, antiidiotype vaccine, dendritic cells vaccine or dna vaccination.In other embodiments, the present invention includes antibody of the present invention and anti-EGFRviii, CD44 splice variant and PSMA vaccine and unite use.The vaccine based on cell that the present invention includes people's descriptions such as antibody of the present invention and Segal is united use (U.S. Patent number 6,403,080 is quoted in full and includes the present invention in).With the vaccine based on cell of the antibody combined use of the present invention can be from body or allochthonous.In brief, the disclosed Theratope of people such as Segal is based on Genitrix, the Opsonokine of LLC (TM) product.Opsonokines ^TMBe genetically engineered cytokine, when mixing, automatically attached to cell surface with tumour cell.When " adorned " cell during as vaccine administration, the cytokine on the cell activates crucial antigen presenting cell in the inoculation human body, makes antigen presenting cell picked-up tumour cell simultaneously.Antigen presenting cell can indicate " killer " T cell to seek and destroy the similar tumour cell of whole body.Therefore, Opsonokine ^TMProduct is transformed into the effective antitumour immunity therapeutic preparation with tumour cell.

In one embodiment, the present invention includes the known any allergy vaccine of humanized antibody of the present invention and prior art and unite use.For example, humanized antibody of the present invention can be united use with the reorganization hybrid molecule of the main thimothy grass pollen allergen of encoding, this molecule is used to the anaphylactoid vaccine of showy flowers of herbaceous plants powder (people such as Linhart, 2000, FASEB Journal, 16 (10): 1301-3 is quoted in full and is included in the present invention).In addition, the dna vaccination that humanized antibody of the present invention can be described with people such as Horner (2002, Allergy, 57Suppl, 72:24-9 is quoted in full and includes the present invention in) is united use.Antibody of the present invention can be united use with downward modulation IgE secretion (people such as Choi, 2002, Ann.Allergy Asthma Immunology, 88 (6): 584-91 with BacilleClamett-Guerin (" BCG ") vaccine; People such as Barlan, 2002, Journal Asthma, 39 (3): 239-46; Two pieces of documents are quoted in full includes the present invention in).Humanized antibody of the present invention can be used for the treatment of food anaphylaxis.Humanization antibody particularly of the present invention can with vaccine or known other immunotherapy of prior art (referring to people such as Hourihane, 2002, Curr, Opin.Allergy Clin.Immunol.2 (3): 227-31) unite use with the anaphylaxis of treatment peanut.

The inventive method and composition can be united use with the vaccine that needs antigen immune.Antigen can be the known any antigen of prior art.For example, humanized antibody of the present invention can be used for strengthening to infectant, disease or paracytic immune response, such as but not limited to: bacterium (as gram positive bacterium, gram negative bacterium, aerophil, spirobacteria, mycobacterium, rickettsia, chlamydozoan etc.), parasite, fungi (as Candida albicans, aspergillus etc.), virus (as dna virus, RNA viruses etc.) or tumour.Virus infection includes but not limited to: human immunodeficiency virus (HIV); Hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus or other hepatitis virus; Cytomegalovirus (CMV), hsv s-1 (2 ,-3 ,-4 ,-5 ,-6), human papillomavirus; Respiratory syncytial virus (RSV), parainfluenza virus (PIV), Epstein-Barr virus, human stroma lung virus (HMPV), influenza virus, severe acute respiratory syndrome (SARS) or any other virus infection.

The present invention includes the vaccine composition and the method that comprise humanized antibody of the present invention, antigen and combination of cytokines.Preferably, described cytokine is IL-4, IL-10 or TGF-β.

The present invention includes and use humanized antibody of the present invention to strengthen body fluid and/or cell-mediated at the antigenic reaction of vaccine composition.The present invention further comprises use antibody prevention of the present invention or treatment specified disease, wherein strengthens and can effectively treat or preventing disease at specific antigen or multiple antigenic immune response.This class disease is including, but not limited to virus infection, as HIV, CMV, hepatitis, simplexvirus, measles etc.; Infectation of bacteria; Fungi and parasitic infection; Cancer; With any can be by strengthening other disease that the immune response of specific antigen is treated or prevented.

5.4 composition and medication

The invention provides the pharmaceutical composition and the method that contain humanized antibody of the present invention.Fusion rotein of the present invention by using significant quantity to individuality or yoke close molecule or contain inventor's fusion rotein or yoke closes the pharmaceutical composition of molecule, and the present invention also provides treatment, prevention and improvement and disease, symptom or infected the method for one or more relevant symptoms.Preferably, antibody or fusion rotein or yoke close (promptly not containing those substantially limits its effect or produce the material of the side effect of not expecting) that molecule is purifying basically.In a specific embodiments, this individuality is an animal, and Mammals preferably is as non-human primate (as ox, pig, horse, cat, dog, mouse etc.) and primates (as the monkey class, for example the stump-tailed macaque and the mankind).In a preferred embodiment, individuality is human.

The known composition that has multiple drug delivery system can be used for containing humanized antibody of the present invention, as liposome methods, microparticle, micro-capsule, the reconstitution cell that can express this antibody or fusion rotein, receptor mediated endocytosis (Wu and Wu, 1987, J.Biol.Chem.262:4429-4432), as constructs of retrovirus or other carrier part etc.

In some embodiments, for target transmits antibody of the present invention, humanized antibody of the present invention is formulated into liposome.Liposome is made of the phospholipid bilayer of entad arranging, and includes water.Liposome generally contains various types of lipids, phosphatide and/or tensio-active agent.The component of liposome is arranged with double-deck configuration, and the lipid that is similar in the microbial film is arranged.Liposome is particularly preferred drug administration carrier, and partly cause is their biocompatibility, reduced immunogenicity and hypotoxicity.Method for preparing lipidosome is that prior art is known and be included in the scope of the present invention, referring to: people such as Epstein, 1985, Proc.Natl.Acad.Sci.USA, 82:3688; People such as Hwang, 1980 Proc.Natl.Acad.Sci.USA, 77:4030-4; Do not have the patent No. 4,485,045 and 4,544,545; All documents are quoted in full includes this paper in.

The present invention comprises that also preparation prolongs the method for the liposome of (being to increase cycling time) plasma half-life, and is as U.S. Patent number 5,013, disclosed in 556.The preferred liposome that the present invention uses can not removed the mononuclear phygocyte system that promptly is not ingested (MPS) fast from circulation.The present invention includes the spatial disposition stabilized liposomes, use the known general method preparation of one of ordinary skill in the art.Although be not intended to be subjected to the restriction of particular mechanism of action, the spatial disposition stabilized liposome contains the lipid composition of the hydrophilic residue of a large amount of highly flexibles, reduced untoward reaction between liposome and the plasma proteins, reduced the opsonization of plasma component and reduced the identification of MPS.The spatial disposition stabilized liposomes is preferably used the polyoxyethylene glycol preparation.About the preparation of liposome and sterically stabilized liposome, referring to: people such as Bendas, 2001 BioDrugs, 15 (4): 215-224; People such as Allen, 1987 FEBS Lett.223:42-6; People such as Klibanov, 1990 FEBS Lett., 268:235-7; People such as Blum, 1990, Biochim.Biophys.Acta., 1029:91-7; People such as Torchilin, 1996, J.Liposome Res.6:99-116; People such as Litzinger, 1994, Biochim.Biophys.Acta, 1190:99-107; People such as Maruyama, 1991, Chem.Pharm.Bull, 39:1620-2; People such as Klibanov, 1991, Biochim Biophys Acta, 1062; 142-8; People such as Allen, 1994, Adv.Drug Deliv.Rev, 13:285-309; All documents are quoted in full includes the present invention in.The present invention also comprises the liposome (referring to U.S. Patent number 4,544,545) of certain organs target or the liposome (referring to Application No. 2005/0074403) of specific cells target.Can use the composition that contains Yelkin TTS, cholesterol and PEG deutero-phosphatidylethanolamine (PEG-PE) to the present composition and the useful especially liposome of method, generate by reverse phase evaporation.Extrude liposome by the definite strainer in aperture and generate liposome with required diameter.In some embodiments, antibody fragment of the present invention as F (ab '), can utilize preceding method and liposome crosslinked, referring to people such as Martin, and 1982, J Biol.Chem.257:286-288 is quoted in full and is included in the present invention.

Humanized antibody of the present invention can also be made into immunoliposome.Immunoliposome is the liposome composition that antibody of the present invention or its fragment covalently or non-covalently are connected the surface.The chemical process that antibody is connected to surface of liposome is that prior art is known and be included in the scope of the invention, referring to: U.S. Patent number 6,787,153; People such as Allen, 1995, Stealth Liposomes, Boca Rotan:CRC Press, 233-44; People such as Hansen, 1995, Biochim.Biophys.Acta, 1239:133-44; All quoted in full and included in the present invention.In the most preferred embodiment, the immunoliposome that is used for the inventive method and composition still is that spatial disposition is stable.Preferably, humanized antibody of the present invention covalently or non-covalently is connected with hydrophobic anchor, and this hydrophobic anchor is by firmly in the double-layer of lipoid of liposome.The example of hydrophobic anchor includes but not limited to phosphatide, as phosphatidylethanolamine (PE), phosphatidylinositols (PI).For realizing the covalently bound of antibody and hydrophobic anchor, can use any known biochemical method in the prior art, referring to: J.Thomas August, ed, 1997, GeneTherapy:Advances in Pharmacology, Volume 40, Academic Press, San Diego, CA., the 399-435 page or leaf is quoted in full and is included in the present invention.For example, active group reaction on the hydrophobic anchor that functional group on the antibody molecule can be connected with liposome for example, activates through water-soluble carbodiimide, the amino of antibody lysine side-chain can be coupled described phosphatidylethanolamine with the liposome that is connected with N-glutaryl-phosphatidylethanolamine; Perhaps pass through for example pyridine sulphur propionyl-phosphatidylethanolamine of sulfydryl reactive group, the sulfydryl of the antibody that is reduced can be coupled with liposome.Referring to: people such as Dietrich, 1996, Biochemistry, 35:1100-1105; People such as Loughrey, 1987, Biochim.Biophys.Acta, 901:157-160; People such as Martin, 1982, J.Biol.Chem.257:286-288; People such as Martin, 1981, Biochemistry, 20:4429-38; All documents are quoted in full includes the present invention in.Although be not intended to be subjected to the restriction of particular mechanism of action, the immunoliposome preparation that contains antibody of the present invention is effective especially therapeutic preparation, because they are sent into antibody in the tenuigenin of target cell, described target cell contains the Fc γ RIIB acceptor with this antibodies.Preferably, this immunoliposome has the transformation period in the blood halflife of prolongation, particularly target cell, can internalization enter in the tenuigenin of target cell, thereby avoids the loss of therapeutic preparation or the degraded of endolysosome approach.

The present invention includes and contain humanized antibody of the present invention or its segmental immunoliposome.In some embodiments, this immunoliposome further comprises one or more other therapeutic preparations, as therapeutic preparation disclosed by the invention.

Immunoliposome composition of the present invention comprises one or more lipids that can form carrier, antibody of the present invention or its fragment or derivative and (optionally) hydrophilic polymer.The lipid that can form carrier is preferably the lipid with two carbohydrate chains (as acyl chain and polar head group).The example that can form the lipid of carrier comprises phosphatide (as Yelkin TTS, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositols, sphingophospholipid) and glycolipid (as cerebroside, ganglioside).Other lipid that is used for preparation of the present invention is that one of ordinary skill in the art are known and be included in the scope of the invention.In some embodiments, described immunoliposome composition further comprises hydrophilic polymer, and as polyoxyethylene glycol and Sphingolipids,sialo GMl, it has prolonged the plasma half-life of this liposome.The method that yoke closes hydrophilic polymer and liposome is well known in the prior art and is included in the scope of the invention.About the summary of immunoliposome and preparation method thereof, referring to: U.S. Patent Application Publication No. 2003/0044407; PCT international publication number WO 97/38731, people such as Vingerhoeads, 1994, Immimomethods, 4:259-72; Maruyama, 2000, Biol.Pharm.Bull.23 (7): 791-799; People such as Abra, 2002, Journal of Liposome Research, 12 (1﹠amp; 2): 1-3; Park, 2002, BioscienceReports, 22 (2): 267-281; People such as Bendas, 2001 BioDrugs, 14 (4): 215-224, J.ThomasAugust, ed, 1997, Gene Therapy:Advances in Pharmacology, Volume 40, AcademicPress, San Diego, CA., 399-435 page or leaf, all documents are quoted in full includes the present invention in.

The medication of humanized antibody of the present invention includes but not limited to: administered parenterally method (as intracutaneous, intramuscular, intraperitoneal, intravenously and subcutaneous), epidural and mucous membrane (for example nose is interior and oral route).In a specific embodiments, antibody of the present invention adopts intramuscular, intravenously or subcutaneous administration.Composition can be applied for example infusion or the injection of bullet formula, epithelium or mucosal absorption (as oral mucosa, rectum and intestinal mucosa etc.) by any conventional route, and can be with other biologically active agent administration.Administration can be a whole body or partial.In addition, can also adopt the lung administration, for example by use sucker or atomizer, and the propellant preparation.Referring to: U.S. Patent number 6,019,968,5,985,20,5,985,309,5,934,272,5,874,064,5,855,913,5,290,540 and 4,880,078; PCT publication number WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346 and WO 99/66903, every piece of patent documentation is all quoted in full includes the present invention in.

The present invention also provides humanized antibody of the present invention has been packaged in the sealed vessel, as the ampoule or the pouch of indication antibody amount.In one embodiment, antibody of the present invention provides with aseptic freeze-dried powder or anhydrous enriching agent in the container of sealing, and can be made into suitable concentration again with for example water or physiological saline solution and be administered to the patient.Preferably, antibody of the present invention provides with aseptic freeze-dried powder or anhydrous enriching agent in the container of sealing, and its unitary dose is at least 5mg, more preferably 10mg, 15mg, 25mg, 35mg, 45mg, 50mg or 75mg at least at least at least at least at least at least at least.Lyophilized antibodies of the present invention is preserved down at temperature 2-8 ℃ in original container, and dissolve again back 12 hours, be preferably in 6 hours, use in 5 hours, in 3 hours or in 1 hour.In a selectable embodiment, antibody of the present invention provides with liquid form in indication contained antibody, fusion rotein or yoke close the sealed vessel of the dosage of molecule and concentration.Preferably, the antibody concentration of liquid form is at least 1mg/ml in the sealed vessel, more preferably 2.5mg/ml, 5mg/ml, 8mg/ml, 10mg/ml, 15mg/ml, 25mg/ml, 50mg/ml, 100mg/ml, 150mg/ml, 200mg/ml at least at least at least at least at least at least at least at least at least at least.

Can determine to treat, prevent or improve the significant quantity of the present composition of one or more disease related symptom by the clinical technology of standard.Accurate dosage in the preparation depends on route of administration and disease severity, should decide according to doctor's judgement and each patient's situation.Can infer significant quantity by amount effect curve external or the animal model experiment system.

For antibody of the present invention, the dosage that gives the patient is generally the 0.0001mg/kg-100mg/kg weight in patients.Preferably, dosage is 0.0001mg/kg-20mg/kg, 0.0001mg/kg-10mg/kg, 0.0001mg/kg-5mg/kg, 0.0001mg/kg-2mg/kg, 0.0001mg/kg-1mg/kg, 0.0001mg/kg-0.75mg/kg, 0.0001mg/kg-0.5mg/kg, 0.0001mg/kg-0.25mg/kg, 0.0001mg/kg-0.15mg/kg, 0.0001mg/kg-0.10mg/kg, 0.001mg/kg-0.5mg/kg, 0.01mg/kg-0.25mg/kg, 0.01mg/kg-0.10mg/kg weight in patients.Usually, owing to the immune response to heterologous polypeptide, human antibodies has the long transformation period with respect to the antibody from other kind in human body.Therefore, normally possible than low dosage and low frequency ground administration of human antibody-like.In addition, can strengthen the absorption and the tissue infiltration of antibody, thereby reduce antibody of the present invention or its segmental dosage and administration frequency by for example modifying fatization etc.

In one embodiment, when using as independent preparation, the antibody dosage of the present invention that gives the patient is 0.01mg-1000mg/ days.In the another one embodiment, antibody of the present invention and other therapeutic composition are united use, and the dosage that gives patient's antibody of the present invention is lower than the dosage that this antibody uses separately.

In a specific embodiments, possibly pharmaceutical composition of the present invention is locally applied to the zone that needs treatment, such administration can be undertaken by following manner, such as but not limited to local infusion, injection or implant, described implant is porous, non-porous or gelatinous material, comprises film (as the sialate film) or fiber.Preferably, when using this paper invention antibody, note using those not adsorb the material of antibody or fusion rotein.

In the another one embodiment, the present composition can by carrier particularly liposome carry (referring to Langer, Science 249:1527-1533 (1990); People such as Treat, in Liposomes in theTherapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, 353-365 page or leaf (1989); Lopez-Berestein, ibid, the 317-327 page or leaf; The same).

In the another one embodiment, composition of the present invention can be carried by controlled release or slow-released system.The known any technology of one of ordinary skill in the art can be used for preparing the sustained release preparation that contains one or more antibody of the present invention, referring to: U.S. Patent number 4,526,938; PCT publication number WO 91/05548; PCT publication number WO 96/20698; People such as Ning, 1996, " Intratumoral Radioimmunotheraphy of aHuman Colon Cancer Xenograft Using a Sustained-Release Gel, " Radiotherapy ﹠amp; Oncology 39:179-189; People such as Song, 1995, " Antibody Mediated Lung Targeting ofLong-Circulating Emulsions, " PDA Technology 50:372-397; People such as Cleek, 1997, " Biodegradable Polymeric Carriers for a bFGF Antibody for CardiovascularApplication, " Pro.Int ' l.Symp.Control.ReI.Bioact.Mater.24:853-854; People such as Lam, 1997, " Microencapsulation of Recombinant Humanized Monoclonal Antibody forLocal Delivery; " Proc.Int ' l.Symp.Control ReI.Bioact.Mater.24:759-760, every piece of document is all quoted in full includes the present invention in.In one embodiment, can in controlled release system, use pump (referring to Langer, supra; Sefton, 1987, CRC Crit.Ref.Biomed.Eng.14:20; People such as Buchwald, 1980, Surgery 88:507; People such as and Saudek, 1989, N.Engl.J.Med.321:574).In the another one embodiment, polymeric material can be used for realizing that the controlled release of antibody is (referring to Medical Applications ofControlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen andBall (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J., Macromol Sci.Rev.Macromol.Chem.23:61; People such as Levy, 1985, Science 228:190; People such as During, 1989, Ann.Neurol.25:351; People such as Howard, 1989, J.Neurosurg.71:105); U.S. Patent number 5,679,377; U.S. Patent number 5,916,597; U.S. Patent number 5,912,015; U.S. Patent number 5,989,463; U.S. Patent number 5,128,326; PCT publication number WO 99/15154; PCT publication number WO 99/20253).The example that is used for the polymkeric substance of sustained release preparation includes but not limited to: poly-(2-hydroxyl ethyl methacrylate), poly-(methyl methacrylate), poly-(vinylformic acid), poly-(ethane-acetic acid ethyenyl ester), poly-(methylacrylic acid), polyglycolic acid ester (PLG), poly-acid anhydrides, poly-(N-V-Pyrol RC), poly-(vinyl alcohol), polyacrylamide, poly-(ethylene glycol), polylactide (PLA), poly-(rac-Lactide-oxyacetic acid) are (PLGA) and poe.In the another one embodiment, controlled release system can be placed in treatment target spot (as lung) near, so the part that only need be administered systemically (referring to: Goodson, in Medical Applications or Controlled Release, as mentioned above, vol.2,115-138 page or leaf (1984)).In the another one embodiment, used polymeric composition, referring to people such as Dunn (United States Patent (USP) 5,945,155) as the controlled release implant.Concrete grammar is based on the result of treatment of drinking the biological active materials of original position sustained release the compound system from poly-.Can implant at the intravital any position of patient of needs treatment.In the another one embodiment, used the slow-released system of non-polymer, thereby the implant of the non-polymer that uses is as drug delivery system in patient's body.In case implant, the organic solvent of this implant will scatter from composition, dissipate or leach, and enter in the surrounding tissue liquid, and non-cohesive material will condense or precipitate formation solid, micropore matrix (U.S.5,888,533) gradually.

Controlled release system has discussion (1990, Science 249:1527-1533) in the summary of Langer.The known any technology of one of ordinary skill in the art can be used to prepare the sustained release preparation that contains one or more therapeutic preparations of the present invention.Referring to for example: U.S. Patent number 4,526,938; International publication number WO 91/05548 and WO 96/20698; People such as Ning, 1996, Radiotherapy ﹠amp; Oncology 39:179-189; People such as Song, 1995, PDA Journal of Pharmaceutical Science ﹠amp; Technology 50:372-397; People such as Cleek, 1997, Pro.Int ' l.Symp.Control.ReI.Bioact.Mater.24:853-854; People such as Lam, 1997, Proc.Int ' l.Symp.Control ReI.Bioact.Mater.24:759-760, every piece of document is all quoted in full includes the present invention in.

In a specific embodiments, the present composition is the nucleic acid of encoding antibody, with the expression of using the antibody that can strengthen its coding in the described nucleic acid body, this administration is to be undertaken by this nucleic acid construct being become the part of suitable expression vector make it to become cellular content, for example use retroviral vector (U.S. Patent number 4,980,286) or direct injection or use microparticle bombardment (as particle gun; Biolistic Dupont), perhaps uses lipid, cell surface receptor or transfection reagent bag quilt, perhaps be connected to the known (people such as Joliot on the nuclear homeobox sample peptide that enters, 1991, Proc.Natl.Acad.Sci.USA88:1864-1868), or the like.Alternative, nucleic acid can be imported into host cell and be incorporated in the host cell DNA and expressed by homologous recombination.

For antibody, be applied to individual treatment or prevent significant quantity to be generally the 0.1mg/kg-200mg/kg whose body weight.Preferably, application dosage is the 0.1mg/kg-20mg/kg weight in patients, more preferably the 0.1mg/kg-10mg/kg whose body weight.By modify lipidization for example strengthen the absorption of antibody or fusion rotein and tissue infiltration (as, enter lung), can reduce the dosage and the administration frequency of antibody of the present invention.

Use the antibody of the present invention of treatment or prevention significant quantity can comprise independent treatment, or preferably include serial therapy the treatment that the patient carries out.In a preferred embodiment, the antibody consumption of the present invention that uses of treatment patient is the 0.1-30mg/kg body weight, and is weekly, lasting about 1-10 week, is preferably 2-8 week, more preferably 3-7 week, 4,5 or 6 weeks more preferably.In other embodiments, pharmaceutical composition of the present invention can be once a day or one day twice or three administrations in a day.In other embodiments, pharmaceutical composition of the present invention can be once in a week, weekly secondary, per two weeks once, every month once, per six weeks once, per February once, 1 year secondary or administration annually.Should be understood that the antibody effective dose that is used for the treatment of can increase or reduce in the particular treatment process.

5.4.1 pharmaceutical composition

Composition of the present invention comprise the bulk drug composition that is used for pharmaceutical compositions (as, impure or unpasteurized composition) and be used to prepare the pharmaceutical composition of unit dosage (as, be fit to the composition of using to individuality or patient).This based composition comprises the preventing and/or treating property preparation disclosed by the invention of prevention or treatment significant quantity, the perhaps combination of these preparations and pharmaceutically acceptable carrier.Preferably, the present composition contains the humanized antibody of the present invention and the pharmaceutically acceptable carrier of prevention or treatment significant quantity.

In a particular, this pharmaceutical composition contains: humanized antibody or its fragment of treatment significant quantity, described antibody or its fragment and Fc γ RIIB bonded avidity greater than with the avidity of Fc γ RIIA; Specificity is in conjunction with the antigenic cytotoxic antibody of cancer; With pharmaceutically acceptable carrier.In another embodiment, described pharmaceutical composition further comprises one or more carcinostatic agents.

In a specific embodiments, term " pharmaceutically acceptable " refers to be used for animal, the particularly mankind's the modulability reagent through federation or state government's approved by management or that be recorded in American Pharmacopeia or other generally acknowledged pharmacopeia.Term " carrier " refers to thinner, adjuvant (as, freund's adjuvant (completely with incomplete)), vehicle or the vehicle in order to the administering therapeutic preparation.This class pharmaceutical carrier can be a sterile liquid, and Ru Shui and oil comprise deriving from oil, animal, plant or synthetic oil, as peanut oil, soya-bean oil, paraffin oil, sesame wet goods.When described pharmaceutical composition during by intravenous injection, water is preferred carrier.Normal saline solution, aqueous dextrose and glycerine solution also can be used as liquid vehicle, particularly injection solution.Suitable drug excipient comprises: starch, glucose, lactose, sucrose, gel, Fructus Hordei Germinatus, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talcum powder, sodium-chlor, exsiccant skimmed milk, glycerine, propylene, ethylene glycol, water, ethanol etc.If desired, said composition can also contain a small amount of wetting agent or emulsifying agent, perhaps the PH buffer reagent.The formulation that these compositions can adopt has solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release preparation etc.

Usually, the composition of the present composition can separately provide or be mixed into unit dosage and provide, and for example, provides with lyophilized powder or anhydrous enriching agent in the sealed vessel (as ampoule or pouch) of lined out activity component content.When composition is given by injection, provide the Injectable sterile water or the physiological saline of ampoule amount, so that composition mixes described composition before using.

Composition of the present invention can be formulated into the form of neutrality or salt.Pharmacy acceptable salt includes but not limited to: with the salt that negatively charged ion forms, deutero-salt such as example hydrochloric acid, phosphoric acid, acetate, oxalic acid, tartrate; With the salt that forms with positively charged ion, as deutero-salt such as sodium, potassium, ammonium, calcium, iron hydroxide, Isopropylamine, triethylamine, 2-ethylaminoethyl alcohol, Histidine, PROCAINE HCL, PHARMA GRADE.

The present invention also provides pharmaceutical composition and the test kit that contains Fc γ RIIB antagonist, is used for preventing, treat, control or improving B cell malignancies or its one or more symptoms.Particularly, the invention provides pharmaceutical composition and the test kit that contains humanization Fc γ RIIB antibody or its Fab.

5.4.2 gene therapy

In a specific embodiments, contain the nucleic acid of antibody or fusion rotein encoding sequence, treat, prevent or improvement and disease, illness or one or more relevant symptoms of infection in mode by gene therapy.Gene therapy refers to treat by the nucleic acid that maybe can express from expression to individuality that use.In embodiments of the invention, this nucleic acid generates the antibody or the fusion rotein of its coding, this antibody or fusion protein mediated treatment or prophylactic effect.

According to the present invention, can use any method of related gene treatment in the prior art.Illustrative methods is described below.

About the generality summary of gene therapy method, referring to people such as Goldspiel, 1993, ClinicalPharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann.Rev.Pharmacol.Toxicol.32:573-596; Mulligan, Science 260:926-932 (1993); Morgan and Anderson, 1993, Ann.Rev.Biochem.62:191-217; May, 1993, TIBTECH 11 (5): 155-215; And Scholl, 2003, J.Biomed Biotechnol 2003:35-47.Operable recombinant DNA technology well known in the prior art: people such as Ausubel, Current Protocols in Molecular Biology, John Wiley ﹠amp; Sons, NY (1993); Kriegler, Gene Transfer and Expression, ALaboratory Manual, Stockton Press, NY (1990).

Aspect preferred, the present composition contains the nucleic acid of encoding antibody, and described nucleic acid is expressed described antibody as the part of expression vector in suitable host.Particularly, this class nucleic acid has promotor, is preferably allogeneic promoter, and this promotor is connected with the antibody coding region operability, described promotor be induction type or composing type, and randomly be tissue-specific.In another embodiment, antibody coding sequence described in the employed nucleic acid molecule and any other required sequence are connected with the zone that homologous recombination takes place at required genomic locus place in promotion at flank, thereby intrachromosomal expression (Koller and the Smithies of described antibody encoding nucleic acid are provided, 1989, Proc.Natl.Acad.ScL USA 86:8932-8935; People such as Zijlstra, 1989, Nature 342:435-438).

The preferred aspect of another one, composition of the present invention contains the nucleic acid of encoding fusion protein, and described nucleic acid is expressed described fusion rotein as the part of expression vector in suitable host.Particularly, this class nucleic acid has promotor, is preferably allogeneic promoter, and this promotor is connected with fusion rotein coding region operability, and described promotor is induction type or composing type, randomly is tissue-specific.In another embodiment, fusion rotein encoding sequence and any other required sequence are connected with the zone of promotion in the required site generation of genome homologous recombination at flank in the employed nucleic acid molecule, thereby the intrachromosomal expression of antibody encoding nucleic acid is provided.

Nucleic acid can directly be conveyed in patient's body, and in this case, described patient directly is exposed to this described nucleic acid or carries the carrier of nucleic acid; Nucleic acid also can be conveyed in patient's body indirectly, in this case, at first at the described nucleic acid transformant of external use, will be implanted in patient's body by cell transformed then.These two kinds of methods all are in the known body or the outer-gene therapy.

In a specific embodiments, nucleotide sequence directly is applied in the body and is expressed the antibody that generates its coding.This can realize by the known several different methods of prior art, thereby the part for example by they being built into suitable nucleic acid expression vector is also used these nucleotide sequences with it and is become cellular content, for example use retrovirus or other virus vector of defective or attenuation to infect (referring to U.S. Patent number 4,980,286), perhaps pass through the direct injection naked DNA, or use microparticle bombardment (as particle gun; Biolistic; Dupont); perhaps with lipid or cell surface receptor or transfection reagent bag quilt; in liposome, particulate or micro-capsule, wrap up; perhaps they are used after nuclear polypeptide is connected with known can entering; perhaps they are connected with part and participate in receptor-mediated endocytosis (referring to Wu and Wu, 1987, J.Biol.Chem.262:4429-4432) cell of those specific expressed acceptors of target (can be used for) etc.In another embodiment, can form nucleic acid-ligand complex, wherein, part contains the fusion viral peptide of the endosome that breaks, and makes nucleic acid avoid being degraded by lysosome.In the another one embodiment, by the targeting specific acceptor, nucleic acid can be directed in vivo and be absorbed by cell-specific and express (referring to U.S. Patent Application Publication No. 2005/0002903; PCT publication number WO 92/06180, WO 92/22635, WO92/20316, WO93/14188, WO 93/20221).Perhaps, by homologous recombination, nucleic acid can be imported in the cell and be incorporated into expresses (Koller and Smithies, 1989, Proc.Natl.Acad.Sci.USA 86:8932-8935 in the host cell DNA; People such as Zijlstra, 1989, Nature 342:435-438).

In a specific embodiments, used the virus vector of the nucleotide sequence that contains encoding antibody or fusion rotein.For example, can use retroviral vector (people such as Miller, 1993, Meth.Enzymol.217:581-599).These retroviral vectors contain correct packaging virus genome and to be integrated into host cell DNA be necessary component.Coding is used for the antibody of gene therapy or the nucleotide sequence of fusion rotein is cloned into one or more carriers, promotes this nucleotide sequence to be transfused in patient's body.Relevant retroviral detailed content has wherein been described the use retrovirus mdr1 gene has been changed in the hemopoietic stem cell referring to people such as Boesen (1994, Biotherapy 6:291-302), and purpose is to make stem cell more can tolerate chemotherapy.The reference that other explanation retrovirus is used for gene therapy has: people such as Clowes, 1994, J.Clin.Invest.93:644-651; People such as Klein, 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; Grossman and Wilson, 1993, Curr.Opin.in Genetics and Devel.3:110-114.

Adenovirus is the another kind of virus vector that can be used for gene therapy.Adenovirus is attracting especially carrier for gene being imported the respiratory epithelium cell.Adenovirus is infect respiratory epithelial cell and cause slight disease naturally.Other target spot of adenovirus drug delivery system is liver, central nervous system, endotheliocyte and muscle.Adenovirus has the advantage that can infect the undifferentiated cell.Kozarsky and Wilson (Current Opinion inGenetics and Development 3:499-503,1993) have described the gene therapy based on adenovirus.People such as Bout (Human Gene Therapy, 5:3-10,1994) have proved and have used adenovirus carrier that gene is changed in the respiratory epithelium cell of rhesus monkey.Other example that adenovirus is used in gene therapy is referring to people such as Rosenfeld, 1991, Science 252:431-434; People such as Rosenfeld, 1992, Cell 68:143-155; People such as Mastrangeli, 1993, J Clin.Invest.91:225-234; PCT publication number WO94/12649; People such as Wang, 1995, Gene Therapy 2:775-783.In preferred embodiments, use adenovirus carrier.

Adeno associated virus (AAV) also has been suggested and has been used for gene therapy (people such as Walsh, 1993, Proc.Soc.Exp.Biol.Med.204:289-300 and U.S. Patent number 5,436,146).

Another mode of gene therapy relate to will be by electroporation, liposome transfection, calcium phosphate mediation transfection or method such as virus infection gene is changed in the tissue culture cells.Usually, method for transformation comprises selected marker is transformed in the cell.Then described cell is selected, separated the cell of those picked-ups and expression transforming gene.Then, these cells are transfused in patient's body.

In this embodiment, with the nucleic acid transfered cell, will use in the reconstitution cell body that obtain then.Above-mentioned importing can adopt the known any method of prior art to carry out, and includes but not limited to: transfection, electroporation, microinjection, with the transgenosis of the transgenosis of the virus that contains this nucleotide sequence or phage vector infection, cytogamy, karyomit(e) mediation, minicell mediation, spheroplast fusion etc.The many technology with the foreign gene transfered cell of being used for be prior art known (referring to for example, Loeffler and Behr, 1993, Meth.Enzymol.217:599-618, people such as Cohen, 1993, Meth.Enzymol.217:618-644; And Clin.Pharma.Ther.29:69-92,1985) and can be used for the present invention, prerequisite is that essential growth of recipient cell and physiological function do not have destroyed.Described technology should be able to nucleic acid stability change in the cell so that this nucleic acid can be expressed by cell, and preferably, this nucleic acid can by cell offspring heredity and expressing.

The reconstitution cell that obtains can be by the known the whole bag of tricks input of prior art patient.Reorganization hemocyte (as hemopoietic stem cell or progenitor cell) is preferably intravenously and uses.Described those skilled in the art are according to the amount of definite required cells such as state of desired effect, patient.

Be imported into the cell that nucleic acid is used for gene therapy and comprise cell type any needs, obtainable, include but not limited to: epithelial cell, endotheliocyte, keratinocyte, inoblast, muscle cell, liver cell; Hemocyte, as T lymphocyte, bone-marrow-derived lymphocyte, monocyte, scavenger cell, neutrophilic leukocyte, eosinophil, megalokaryocyte, granulocyte, various stem cell or progenitor cell, particularly hemopoietic stem cell or progenitor cell are as the stem cell from marrow, cord blood, peripheral blood, fetus liver etc.

In a preferred embodiment, the cell that is used for gene therapy is patient's a autogenous cell.

Be used for an embodiment of gene therapy at reconstitution cell, the nucleotide sequence of encoding antibody or fusion rotein is imported into cell, and described nucleotide sequence can be expressed by this cell or cell offspring, the gained reconstitution cell is applied in the body treats subsequently.In a specific embodiments, stem cell or progenitor cell have been used.Can all may be used for embodiment of the present invention (referring to PCT publication number WO 94/08598 at in-vitro separation and any stem cell of cultivating and/or progenitor cell; Stemple and Anderson, 1992, Cell71:973-985; Rheinwald, 1980, Meth.Cell Bio.21 A:229; Pittelkow and Scott, 1986, Mayo Clinic Proc.61:771).

In a specific embodiments, the nucleic acid that imports for the gene therapy purpose contains the inducible promoter that is connected in the coding region by operability, thereby by controlling the suitable existence of transcribing inductor or not existing, controls described expression of nucleic acids.

5.4.3 test kit

The invention provides pharmaceutical pack or test kit, it contains one or more containers that humanized antibody of the present invention is housed.In addition, can also comprise in described pharmaceutical pack or the test kit one or more other be used for the treatment of the preventative preparation or the therapeutic preparation of disease.The present invention also provides pharmaceutical pack or test kit, and it comprises one or more containers, and one or more compositions of pharmaceutical composition of the present invention are housed in the container.Randomly, these containers are with specification sheets, and its form meets the regulation of the government department of production, use or the sale of managing medicine or biological products, show that obtaining production, use or sales department's approval is used for the mankind.

The invention provides the test kit that can be used for aforesaid method.In one embodiment, this test kit comprises one or more humanized antibodies of the present invention.In the another one embodiment, this test kit further comprises one or more containers, and other preventative preparation or therapeutic preparation that one or more are used for the treatment of cancer therapy are housed in it.In the another one embodiment, this test kit further contains and one or more one or more cytotoxic antibodies of cancer associated antigens bonded.In certain embodiments, described other preventative preparation or therapeutic preparation are chemotheraping preparations.In other embodiments, described prevention or therapeutic preparation are biology or hormonotherapy preparation.

5.5 sign and proof that treatment is used

Before pharmaceutical composition of the present invention or prevention or therapeutic preparation are used to the mankind, preferably detect several aspects of said composition or preparation earlier, for example detect in as cell culture system external, in vivo as (as the rodent models system) detection in animal model tissue, detect desired therapeutic activity then.For example, can be used for determining whether that the test that gives concrete pharmaceutical composition comprises cell cultures, wherein patient tissue samples is incubated in the nutrient solution and is exposed to or contacts with pharmaceutical composition, observe the effect of said composition, form as the colony formation or the tubulose network in three dimensional matrix film or extracellular matrix that suppress growth and/or reduce in the soft agar to tissue sample.Can obtain tissue sample from the patient by examination of living tissue.This detection can be discerned the most effective prevention or treatment molecule in treatment at each patient.Alternative in addition, be used for substituting the cell of cultivating the patient, can also use tumour cell or malignant cell is to screen therapeutic preparation and method.In a plurality of specific embodiments, can use the representative cell (as the T cell) of the cell type that participates in autoimmunization or inflammatory diseases to carry out vitro detection, whether this class cell is had the effect of expection to determine pharmaceutical composition of the present invention.Many examination criterias of the prior art can be used for estimating this survival and/or growth, for example, and can be by detecting ³The mixing of H thymidine, directly cell counting, detect known and (change as the transcriptional activity of proto-oncogene (as fos, myc) or cell cycle sign) and to detect cell proliferation; Can dye by trypan blue and estimate cell viability; Can form that colony reduces based on morphological change, growth and/or in soft agar or three dimensional matrix film or extracellular matrix in form the minimizing of tubulose network and wait the differentiation of estimating cell.Other detection method comprises raft dependency well known in the prior art, CDC, ADCC and apoptosis experiment.

Before being applied to the mankind, can in suitable animal model system, detect the combination of preventative preparation and/or therapeutic preparation.Described animal model system includes but not limited to: rat, mouse, chicken, ox, monkey, pig, dog, rabbit etc.Can use the known any animal system of prior art.In a specific embodiments of the present invention, in mouse model system, detect the combination of preventative preparation and/or therapeutic preparation.This model system is widely used, and is known to one of ordinary skill in the art.Preventative preparation and/or therapeutic preparation can the administrations of being repeated property.Several aspects of this process can change, and for example, give the tentative plan of preventative preparation and/or therapeutic preparation, and these preparations are individually dosed or the mixing administration.

The preferred animal model that is used for the inventive method is, for example, expresses the transgenic mice of Fc γ R on mouse effector cell, as U.S. Patent number 5,877, and any mouse model of describing in 396 (quoted include the present invention in by the integral body).The transgenic mice that is used for the inventive method includes but not limited to: carry human Fc gamma RIIIA mouse, carry human Fc gamma RIIA mouse, carry the mouse of human Fc gamma RIIB and human Fc gamma RIIIA, the mouse of carrying human Fc gamma RIIB and human Fc gamma RIIA.

After preventative preparation of the present invention and/or therapeutic preparation detect through animal model, can in clinical experiment, detect to determine their effectiveness.Can implement clinical experiment according to the known methodology of one of ordinary skill in the art, and determine best dosage and the approach and the toxicity situation of the present composition with normal experiment.

The anti-inflammatory activity of combination treatment of the present invention can use the various experimental animal models of the known inflammatory arthritis of prior art to measure, referring to: Crofford L.J. and Wilder R.L., " Arthritis andAutoimmunity in Animals "; Arthritis and Allied Conditions:A Textbook ofRheumatology, people such as McCarty (eds.), the 30th chapter (Lee and Febiger, 1993).Can also use the experimental or spontaneous animal model of inflammatory arthritis and autoimmunity rheumatism to estimate the anti-inflammatory activity of combination treatment of the present invention.Mode with non-limiting example provides some detection methods below.

Known and the sacroiliitis of widespread use of prior art or the main animal model of inflammatory diseases comprise: adjuvant inductive rat model of arthritis, collagen-induced rat model of arthritis and mouse model, the rats with arthritis of antigen induction, rabbit and hamster model, referring to: Crofford LJ. and Wilder R.L., " Arthritis andAutoimmunity in Animals "; Arthritis and Allied Conditions:A Textbook ofRheumatology, people such as McCarty (eds.), the 30th chapter Lee and Febiger, 1993), quoted in full and included in the present invention.

The anti-inflammatory activity of combination treatment of the present invention can use carrageenin inductive rat model of arthritis to estimate.Carrageenin inductive sacroiliitis also has been applied to studying in rabbit, dog and the pig chronic arthritis or inflammation.Be used to monitoring therapeuticing effect with the quantitative tissue morphometry.Use the method for this carrageenin inductive arthritis model, referring to people such as Hansra P., " Carrageenan-induced Arthritis in theRat, " Inflammation, 24 (2): 141-155, (2000).Known and the known zymosan inductive of prior art inflammatory animal model in addition commonly used.

Can also estimate the anti-inflammatory activity of combination treatment of the present invention by the inhibition that detects on Carrageenan inductive rat pawl oedema, use as people " Carrageenan-Induced Edema in Hind Pawof the Rat as an Assay for Anti-inflammatory Drugs " Proc.Soc.Exp.Biol Med.Ill such as Winter C.A., 544-547, improving one's methods described in (1962).This detection method has become sieve method in the main body that detects most of non-steroidal anti-inflammatory drugs (NSAID) anti-inflammatory activity, and is considered to predict the result of treatment to the people.The anti-inflammatory activity of prevention or therapeutic preparation shows as with respect to carrier administration control group, to the inhibition percentage ratio of experimental group rat hind paw weight increase.

In addition, the animal model of inflammatory bowel also can be used for estimating effect (people such as Kim, 1992, the Scand.J.Gastroentrol.27:529-537 of combined therapy of the present invention; Strober, 1985, Dig.Dis.Sci.30 (12Suppl): 35-10S).Ulcerative colitis and Crohn disease disease are the human inflammatory bowels of inductive in animal body.Can give the oral sulfated polysaccharide of animal and include but not limited to that amylopectin, carrageen, Amylopectin Sultate and T 500 or chemical irritant (including but not limited to trinitrobenzene sulfuric acid (TNBS) and acetate) induce inflammatory bowel.

Animal model in asthma also can be used for estimating the effect of combination treatment of the present invention.An example of this class model is mouse adoptive transfer model (adoptive transfer model), wherein the aeroallergen of TH1 or TH2 acceptor mouse causes TH effector cell to migrate to air flue, and be attended by intensive neutrophilic granulocyte (TH1) and eosinophilic granulocyte (TH2) lung mucosal inflammatory reactivity (people such as Cohn, 1997, J Exp.Med.1861737-1747).

The animal model of autoimmune disorder also can be used for estimating the effect of combination treatment of the present invention.Set up the animal model of autoimmune disorder, as type i diabetes, Tiroidina autoimmune disorder, systemic lupus erythematosus and brightic model (people such as Flanders, 1999, Autoimmunity1^.HS-1A β; People such as Krogh, 1999, Biochimie 81:511-515; Foster, 1999, Semin.Nephrol.19:12-24).

In addition, the known any detection method of described those skilled in the art can both be used for estimating the prevent and/or treat effectiveness of combination treatment disclosed by the invention to autoimmunization and/or inflammatory diseases.

Can learn method by standard drug and in cell culture or laboratory animal, detect virulence and the effectiveness that the present invention prevents and/or treats scheme, for example, measure LD ₅₀(mld) and ED ₅₀(median effective dose).The dosage ratio of toxicity and result of treatment is a therapeutic index, can be expressed as LD ₅₀/ ED ₅₀The preventing and/or treating property preparation that shows high therapeutic index is preferred.Preventing and/or treating property preparation with toxic side effects can be used, but the design drug delivery system of being careful makes said preparation target damaged tissue position, reducing the damage to non-infected cells as far as possible, thereby reduces side effect.

The data that obtain from cell culture experiments and zooscopy can be used for determining that preventing and/or treating property preparation is used for human dosage range.The dosage of described preparation is preferably and comprises ED ₅₀And not or have in the less toxic circulation composition scope.Dosage can change in described scope according to the formulation that adopts and route of administration.The treatment significant quantity of any preparation that uses in the inventive method can be by the cell culture experiments quilt according to a preliminary estimate.Can in animal model, determine to obtain the IC that comprises that cell cultures is measured by dosage ₅₀The circulating plasma concentration range of (that is, reaching the test-compound concentration that the maximum symptom of half suppresses).These information can be used to be identified for more accurately human dosage.Plasma concentration can be detected, for example passes through high performance liquid chromatography.

The antitumour activity of the treatment reagent that the present invention adopts also can detect by the various experimental animal models models that are used for cancer research, as the SCID mouse model or have transgenic mice or the nude mice of human heterograft, known and the disclosed animal model of prior art such as hamster, rabbit etc., referring to: Relevance ofTumor Models for Anticancer Drug Development (1999, eds.Fiebig and Burger); Contributions to Oncology (1999, Karger); The Nude Mouse in Oncology Research (1991, eds.Boven and Winograd); Anticancer Drug Development Guide (1997 ed.Teicher); These documents are quoted by integral body and are included the present invention in.

Before being applied to the mankind, the present invention program and composition are preferably in vitro detection, the treatment or the prophylactic activity of detection of desired in the body then.Can use tumour or malignant cell is to screen therapeutic preparation and method.Multiple examination criteria of the prior art can be used for estimating this survival and/or growth; For example, can be by detecting ³The mixing of H thymidine, directly cell counting, (cell proliferation is measured in the variation as proto-oncogene (as fos, myc) or cell cycle sign) transcriptional activity to detect known; Can dye by trypan blue and estimate the vigor of cell; Can change, grow based on form slow down and/or soft agar in the minimizing that forms of colony or the minimizing that forms of the tubulose network in three dimensional matrix film or the extracellular matrix wait evaluate differentiation.

Before human detection, can in suitable animal model system, detect the compound that is used for the treatment of, this system includes but not limited to rat, mouse, chicken, ox, monkey, rabbit, hamster etc., and animal model for example mentioned above.Then, described compound can be applied in the suitable clinical experiment.

In addition, the known any detection method of one of ordinary skill in the art can both be used for estimating combination treatment disclosed by the invention and prevent and/or treat effectiveness in treatment or preventing cancer, inflammatory diseases or autoimmune disease.

5.6 diagnostic method

The antibody of mark of the present invention can be used to diagnostic purpose, detects, diagnosis or monitoring of diseases, illness or infection.The invention provides detection or diagnosis to disease, illness or infection, particularly autoimmune disease, comprise: (a) use the antibody of one or more and Fc γ RIIB immunity specific combination, detect the expression of Fc γ RIIB in patient's cell or tissue sample; (b) compare antigen levels and control level,, tried antigen levels and indicated disease, illness or infection with respect to the increase of contrast antigen levels as the level in the healthy tissues sample.

The immunohistology method of using the present invention disclosed or known classics of one of ordinary skill in the art is (referring to people such as Jalkanen, 1985, J.Cell.Biol.101:976-985; People such as jalkanen, 1987, J Cell.Biol105:3087-3096), can use the Fc γ RIIB level in the antibody test biological sample of the present invention.Other method based on antibody that is used to detect protein gene expression comprises immunodetection, as euzymelinked immunosorbent assay (ELISA) (ELISA) and radioimmunology (RIA).Suitable antibody test mark is that prior art is known, comprising: enzyme labelling, as alkaline phosphatase, glucose oxidase; Radio isotope, as iodine ( ¹²⁵I, ¹³¹I), carbon ( ¹⁴C), sulphur ( ³⁵S), tritium ( ³H), indium ( ¹²¹In) and technetium ( ⁹⁹Tc); Luminescent marking is as luminol,3-aminophthalic acid cyclic hydrazide; Fluorescent mark is as fluorescein and rhodamine.

One aspect of the present invention is disease, illness or the infection in detection and the diagnosis human body.In one embodiment, diagnosis comprises: a) for the patient use (as parenteral, subcutaneous or intraperitoneal administration) significant quantity, with the antibody of Fc γ RIIB immunologic opsonin bonded mark; B) wait for a period of time so that traget antibody preferably concentrates on the position (and making unconjugated tagged molecule be scavenged into background level) of patient's expression in vivo Fc γ RIIB after using; C) measure background level; And d) detects the intravital traget antibody of patient, detect the existence that the traget antibody that is higher than background level then means disease, illness or infection.According to this embodiment, use the imaging group to come traget antibody, described imaging group can use the known imaging system of one of ordinary skill in the art to detect.The substrate level can be measured by several different methods, comprises the amount of detected tagged molecule is compared with the standard value of measuring for particular system before.

This field technique personnel should be appreciated that the imaging system of individual size and use will determine to produce the consumption of the needed imaging group of diagnosis imaging.In the example of radio isotope group, for the human body individuality, radioactive amount of injection is generally about 5-20 millicurie ⁹⁹Tc.Traget antibody will preferentially accumulate in the cell position of containing differential protein.The in-vivo tumour imaging, referring to: people such as S.W.Burchiel, " Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments. " (the 13rd chapter of Tumor Imaging:The Radiochemical Detection of Cancer, S.W.Burchieland B.A.Rhodes, eds., Masson Publishing Inc. (1982).

Comprise the labeling pattern and the method for application of use according to some variablees, in order to make tagged molecule preferentially concentrate in patient's body the position and to make unmarked molecule be scavenged into the substrate level, using the back timed interval is 6-48 hour or 6-24 hour or 6-24 hour.In the another one embodiment, the timed interval after using is 5-20 days or 5-10 days.

In one embodiment, the monitoring of disease, illness or infection can by repeat to diagnose the illness, the method for illness or infection carries out, and for example, repeats diagnosis after after the initial diagnosis 1 month, 6 months, 1 year.

The existence of patient's body internal labeling molecule can use the known method that is used for the body interscan of prior art to detect.These methods are based on the labeling pattern of using.Those skilled in the art can determine that appropriate means detects specific markers.Operable method or equipment include but not limited in the diagnostic method of the present invention: computerized tomography (CT), body scan (as PET (positron emission tomography) (PET)), nuclear magnetic resonance (MRI) and ultrasonic examination.

In a specific embodiments, use the labelled with radioisotope molecule, and use the radiation reaction surgical device in the patient, to detect people such as (, United States Patent (USP) 5,441,050) Thurston.In the another one embodiment, use the fluorescent chemicals tagged molecule, and use the fluorescent reaction scanner in patient's body, to detect.In the another one embodiment, use the positron emitting metal tagged molecule, and use positron emission tomography in patient's body, to detect.In the another one embodiment, use the spin labeling molecule, and use nuclear magnetic resonance (MRI) in patient's body, to detect.

6. embodiment

6.1 the humanization of mouse anti CD32B monoclonal antibody 2B6

RNA is converted to cDNA, and (Ambion Inc.) has carried out pcr amplification to VH and VL fragment to use the RLM-RACE test kit.The gene-specific primer of VH is SJ15R, SEQ ID NO.47 (5 ' GGTCAC TGT CAC TGG CTC AGG G 3 ') and SJ16R, SEQ ID NO.48 (5 ' AGG CGGATC CAG GGG CCA GTG GAT AGA C 3 ').The gene-specific primer of VL is SJ17R, SEQ ID NO.49 (5 ' GCA CAC GAC TGA GGC ACC TCC AGA TG 3 ') and SJ18R, SEQ ID NO.50 (5 ' CGG CGG ATC CGA TGG ATA CAG TTG GTG CAG CATC 3 ').(Invitrogen Inc.) inserts the RACE product among the plasmid pCR2.1-TOPO to use TOPO TA Cloning test kit.The plasmid that generates is carried out VH and the VL sequence that dna sequencing is measured 2B6.The nucleotide sequence that generates is translated and measures the aminoacid sequence of each sequence prediction.From these sequences, determine framework region (FR) and complementary determining region (CDR) with Kabat.Mouse VH combines with people C-Gammal constant region and Ig homing sequence and is inserted into pCI-neo, is used for Mammals and expresses.Mouse VL combines with people C-kappa fragment and Ig homing sequence and is cloned among the pCI-neo, is used for Mammals and expresses.

Humanization 2B6VH consists of: from the FR fragment of human VH fragment VH1-18 and JH6 and the CDR zone of 2B6VH.Humanization 2B6VL consists of: from the FR fragment of human VL fragment VK-A26 and JK4 and the CDR zone of 2B6VL.Humanized VH and VL fragment begin assembling from the oligonucleotide of PCR combination, amplification.By PCR the fragment that generates is combined with homing sequence, the constant region fragment that is fit to is cloned into expression vector pCI-neo as the NheI-EcoRI fragment.Confirm the dna sequence dna of gained plasmid by sequential analysis.For VL, the analyzed plasmid of none has right-on sequence.Reduce the quantity in wrong site in conjunction with two best insertions, correct these wrong sites by rite-directed mutagenesis then.Like this, the light chain segments with predetermined human 2B6VL sequence has obtained evaluation.

Contrasting shown in Figure 1A of the aminoacid sequence of mouse 2B6VH, humanization 2B6VH1-18 and people JH6.Figure 1B has shown mouse 2B6VL, people 2B6VL-1, people 2B6VL-2, people 2B6VL-3 and people The contrasting of aminoacid sequence.

6.2 the expression of humanization 2B6 heavy chain and light chain and sign

Experiment 1:hu2B6 heavy chain (HC) expression plasmid is gone in the HEK-293 cell by cotransfection with ch2B6 light chain (LC).Simultaneously, ch2B6HC and ch2B6LC are by cotransfection.Cultivate after 3 days the amount of the human IgG of being expressed by the ELISA detection by quantitative.Combining by ELISA measuring and solubility Fc γ RIIb-Fc dipolymer then.

The ELISA experimental program: the solubility Fc γ RIIb-Fc in 2.5ng/ hole is caught by mouse anti FcVRIIb antibody 3H7 on 96 hole Maxisorp plates, room temperature 1 hour.Ch2B6 or hu2B6Hc/Ch2B6Lc conditioned medium are added into each plate hole after a series of 2 times of dilutions of 25ng/ aperture.Orifice plate was at room temperature hatched 1 hour, then the F (ab ') that joins by the HRP coupling ₂The anti-IgG F of goat (ab) ' ₂Specific secondary antibody detects combination.Hatch about 45 minutes with secondary antibody after, use TMB matrix to make dull and stereotyped the development.After hatching 5 minutes, use 1%H ₂SO ₄Termination reaction.Under OD450nm, read plate by the SOFTmax program.Between each step, wash orifice plate 3 times with the PBS/0.1% polysorbas20.Before adding solubility Fc γ RIIb-Fc, use the PBS/0.1% polysorbas20 that contains 0.5%BSA to dull and stereotyped room temperature sealing 30 minutes.

The result: the ELISA experimental result shows that the hu2B6HC/ch2B6LC monoclonal antibody is similar to the avidity of receptors bind to the ch2B6HC/ch2B6LC monoclonal antibody to the avidity of receptors bind as shown in Figure 2.

Carry out facs analysis then and detect combining of monoclonal antibody and Daudi cell

The scheme of facs analysis: about 10 ⁶The Daudi cell is used for each antibody staining.Cell through the PBS washing once.Primary antibody (Ch2B6, Hu2B6Hc/ch2B6Lc, human IgG1) is diluted into 0.5,0.1,0.02 μ g/mL in PBS/1%BSA, change 100 μ l dilution antibody over to cell.After hatching 30 minutes under 4 ℃, once with 1ml PBS/1%BSA washed cell.The anti-IgG Fc of the goat specificity F (ab ') that PE is crosslinked ₂(Jackson ImmunoReseach Inc.) uses with dilution in 1: 1000 as secondary antibody fragment.After hatching 30 minutes under 4 ℃, once with 1ml PBS/1%BSA washed cell.Cell is suspended among the PBS/1%BSA of 500 μ l, carries out facs analysis.

The result: the result shows hu2B6HC/ch2B6LC monoclonal antibody and this mankind B cell tumour clone bonded avidity identical with chimeric mAb (table 6).

Table 6:

Main antibody	Concentration (ug/ml)	Mean fluorecence
Main antibody	Concentration (ug/ml)	Mean fluorecence	IgG
1	0.5	9.49
	0.5	9.49		0.1	N/A
	0.02	N/A		0.1	N/A
	0.02	N/A	Ch2B6	0.5	647.48
0.1	511.85			0.5	647.48
0.1	511.85	0.02		172.68
Hu2B6Hc/Ch2B6Lc	0.5	0.02		172.68	648.99
	0.5	0.1	546.46		648.99
	0.02	0.1	546.46	196.93

Experiment 2: use following combination to carry out the transfection of HEK-293 cell: hu2B6HC/hu2B6LC, hu2B6HC/ch2B6LC, ch2B6HC/hu2B6LC and ch2B6HC/ch2B6LC.Cultivate after 3 days, use the amount of such scheme by the expressed human IgG of ELISA detection by quantitative.Measure and dimeric combination of solubility Fc γ RIIb-Fc by ELISA then.This experimental result shows that all monoclonal antibodies combine with similar avidity with acceptor as shown in Figure 3.Use such scheme to carry out facs analysis, detect combine (table 7) of monoclonal antibody and Daudi cell.The result shows that hu2B6HC/hu2B6LC is that bonded avidity is identical with the ch2B6 monoclonal antibody with this mankind B cell tumour.

Table 7:

Main antibody	Concentration (ug/ml)	Mean fluorecence
Main antibody	Concentration (ug/ml)	Mean fluorecence	IgG
1	0.5	6.07
	0.5	6.07		0.1	N/A
	0.02	N/A		0.1	N/A
	0.02	N/A	Ch2B6	0.5	551.52
0.1	514.69			0.5	551.52
0.1	514.69	0.02		168.17
Hu2B6	0.5	0.02		168.17	628.82
	0.5	0.1	618.13		628.82

0.02

228.74

6.3 the generation of HU2B6LC variant, expression and combination

At Hu2B6LC CDR2 district (Asn ₅₀-Val-Ser) common sequences of a N glycosylation site (Asn-Xaa-Ser/Thr) arranged.Thereby be the glycosylation in removing residue 50 sites and the immunogenicity in variation of restriction product and the pharmacology application, the use orthomutation is with position other aminoacid replacement of 50 usefulness (Stratagene test kit).Two kinds of multi-form being generated of Hu2B6LC, Hu2B6LC-N50Y, Hu2B6LC-N50Y, V51Y.Select these amino acid to be because tyrosine is human receptor's residue of CDRL2 position 50 and L-Ala is the residue of CDRL2 position 51 in the human pedigree gene fragment.

Use following combination that the HEK-293 cell is carried out transfection: hu2B6HC/hu2B6LC; Hu2B6HC/hu2B6LC (N50Y); Hu2B6HC/hu2B6LC (N50Y, V51A); Ch2B6HC/ch2B6LC.Cultivate after 3 days, use the amount of the human IgG that aforesaid method expressed by the ELISA detection by quantitative.Measure and dimeric combination of solubility Fc γ RIIb-Fc by ELISA then.This experimental result shows that all monoclonal antibodies are similar to the avidity of receptors bind as shown in Figure 4.Carry out facs analysis, detect combine (table 8) of monoclonal antibody and Daudi cell.The result shows that two variants of hu2B6LC/hu2B6HC monoclonal antibody are that bonded avidity is identical with the ch2B6 monoclonal antibody with this mankind B cell tumour.

Table 8

Main antibody	Concentration (ug/ml)	Mean fluorecence
Main antibody	Concentration (ug/ml)	Mean fluorecence	IgG
1	0.5	1.23
	0.5	1.23		0.1	N/A
	0.02	N/A		0.1	N/A
	0.02	N/A	Ch2B6	0.5	192.88
0.1	141.01			0.5	192.88

	0.02	45.59
	0.02	45.59	Hu2B6	0.5	201.69
0.1	174.37			0.5	201.69
0.1	174.37	0.02		58.65
hu2B6 N50Y	0.5	0.02		58.65	191.16
	0.5	0.1	134.56		191.16
	0.02	0.1	134.56	40.14
	0.02	hu2B6 N50Y，V51A	0.5	40.14	167.16
0.1	133.83		0.5		167.16
0.1	133.83		0.02	45.95

6.4 monoclonal antibody combines with FcRIIA's

The ELISA experimental program: on 96 hole Maxisorp plates, the solubility Fc γ IIA in 100ng/ hole is 40 ℃ of following coated spending the night in carbonate buffer solution.(N50Y, V51A) the IV.3 conditioned medium with purifying begins to be added into each plate hole after a series of 2 times of dilutions from the 25ng/ hole for ch2B6, hu2B6Hc/Ch2B6Lc, hu2B6HC/hu2B6LC (N50Y), hu2B6HC/hu2B6LC.Orifice plate was at room temperature hatched 1 hour.Use the crosslinked F (ab ') of HRP at Ch2B6 and all hu2B6 samples ₂The anti-IgG F of goat (ab) ' ₂Specific secondary antibody and at the crosslinked F of the HRP of IV.3 (ab ') ₂Goat anti-mouse IgG (H+L) secondary antibody detects combination.Hatch about 45 minutes with secondary antibody after, use TMB matrix to make dull and stereotyped the development.After hatching 5 minutes, use 1%H ₂SO ₄Termination reaction.Under OD450nm, read plate by the SOFTmax program.Between each step, wash orifice plate 3 times with the PBS/0.1% polysorbas20.Before the antibody that adds serial dilution, use the PBS/0.1% polysorbas20 that contains 0.5%BSA to dull and stereotyped room temperature sealing 30 minutes.

The result: data presentation, humanized 2B6 antibody does not lose the ability (Fig. 5) of selective binding CD32B in the humanization process.In a word, IV.3 (mouse monoclonal antibody of a kind of anti-Fc γ IIA) is in conjunction with Fc γ IIA, and chimeric and humanized 2B6 debond Fc γ IIA.

The present invention is not intended to be limited in the described scope of specific embodiments, and specific embodiments is the independent description of the indivedual aspects of the present invention, and the method and the component that are equal on the function are also included within the scope of the invention.In fact, except content disclosed by the invention, according to above description and accompanying drawing, various improvement of the present invention are conspicuous to one of ordinary skill in the art.It also is claim of the present invention scope required for protection that this class is improved.

The disclosed full content of various reference that this paper quotes is included into the present invention.

Sequence table

＜110〉Macrogenics Inc.

＜120〉humanization Fc γ RIIB specific antibody and using method thereof

<130>11183-018-228

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<211>17

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Gly

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr

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Glu Lys Val Thr Ile Thr Cys

20

<210>14

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Leu Thr Ile Asn Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

20 25 30

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＜213〉homo sapiens

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1 5 10

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＜223〉humanization 2B6 variable region of light chain-Hu2B6VL-1

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gaaattgtgc tgactcagtc tccagacttt cagtctgtga ctccaaagga gaaagtcacc 60

atcacctgca ggaccagtca gagcattggc acaaacatac actggtacca geagaaacca 120

gatcagtctc caaagctcct catcaagaat gtttctgagt ctatctctgg agtcccatcg 180

aggttcagtg gcagtggatc tgggacagat ttcaccctca ccatcaatag cctggaagct 240

gaagatgctg caacgtatta ctgtcaacaa agtaatacct ggccgttcac gttcggcgga 300

gggaccaagg tggagatcaa a 321

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＜223〉humanization 2B6 variable region of light chain-Hu2B6VL-1

<400>18

Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile

35 40 45

Lys Asn Val Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Thr Trp Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>19

<211>321

<212>DNA

＜213〉artificial sequence

<220>

＜223〉humanization 2B6 variable region of light chain-Hu2B6VL-2

<400>19

gaaattgtgc tgactcagtc tccagacttt cagtctgtga ctccaaagga gaaagtcacc 60

atcacctgca ggaccagtca gagcattggc acaaacatac actggtacca gcagaaacca 120

gatcagtctc caaagctcct catcaagtat gtttctgagt ctatctctgg agtcccatcg 180

aggttcagtg gcagtggatc tgggacagat ttcaccctca ccatcaatag cctggaagct 240

gaagatgctg caacgtatta ctgtcaacaa agtaatacct ggccgttcac gttcggcgga 300

gggaccaagg tggagatcaa a 321

<210>20

<211>107

<212>PRT

＜213〉artificial sequence

<220>

＜223〉humanization 2B6 variable region of light chain-Hu2B6VL-2

<400>20

Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile

35 40 45

Lys Tyr Val Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Thr Trp Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>21

<211>321

<212>DNA

＜213〉artificial sequence

<220>

＜223〉humanization 2B6 variable region of light chain-Hu2B6VL-3

<400>21

gaaattgtgc tgactcagtc tccagacttt cagtctgtga ctccaaagga gaaagtcacc 60

atcacctgca ggaccagtca gagcattggc acaaacatac actggtacca gcagaaacca 120

gatcagtctc caaagctcct catcaagtat gcttctgagt ctatctctgg agtcccatcg 180

aggttcagtg gcagtggatc tgggacagat ttcaccctca ccatcaatag cctggaagct 240

gaagatgctg caacgtatta ctgtcaacaa agtaatacct ggccgttcac gttcggcgga 300

gggaccaagg tggagatcaa a 321

<210>22

<211>107

<212>PRT

＜213〉artificial sequence

<220>

＜223〉humanization 2B6 variable region of light chain-Hu2B6VL-3

<400>22

Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Thr Trp Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>23

<211>363

<212>DNA

＜213〉artificial sequence

<220>

＜223〉humanization variable region of heavy chain-Hu2B6VH-1

<400>23

caggttcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60

tcctgcaagg cttctggtta cacctttacc aactactgga tacactgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggagtg attgatcctt ctgatactta tccaaattac 180

aataaaaagt tcaagggcag agtcaccatg accacagaca catccacgag cacagcctac 240

atggagctga ggagcctgag atctgacgac acggccgtgt attactgtgc gagaaacggt 300

gattccgatt attactctgg tatggactac tgggggcaag ggaccacggt caccgtctcc 360

tca 363

<210>24

<211>121

<212>PRT

＜213〉artificial sequence

<220>

＜223〉humanization variable region of heavy chain

<400>24

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Scr Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asp Pro Ser Asp Thr Tyr Pro Asn Tyr Asn Lys Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Gly Asp Ser Asp Tyr Tyr Ser Gly Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210>25

<211>321

<212>DNA

＜213〉Mustella

<220>

＜223〉mouse 2B6 variable region of light chain

<400>25

gacatcttgc tgactcagtc tccagccatc ctgtctgtga gtccaggaga gagagtcagt 60

ttttcctgca ggaccagtca gagcattggc acaaacatac actggtatca gcaaagaaca 120

aatggttttc caaggcttct cataaagaat gtttctgagt ctatctctgg gatcccttcc 180

aggtttagtg gcagtggatc agggacagat tttattctta gcatcaacag tgtggagtct 240

gaagatattg cagattatta ttgtcaacaa agtaatacct ggccgttcac gttcggaggg 300

gggaccaagc tggaaataaa a 321

<210>26

<211>107

<212>PRT

＜213〉Mustella

<220>

＜223〉mouse 2B6 variable region of light chain

<400>26

Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Thr Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Phe Pro Arg Leu Leu Ile

35 40 45

Lys Asn Val Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Ile Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser Asn Thr Trp Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210>27

<211>363

<212>DNA

＜213〉Mustella

<220>

＜223〉mouse 2B6 variable region of heavy chain

<400>27

caggtccaat tgcagcagcc tgtgactgag ctggtgaggc cgggggcttc agtgatgttg 60

tcctgcaagg cttctgacta ccccttcacc aactactgga tacactgggt aaagcagagg 120

cctggacaag gcctggagtg gatcggagtg attgatcctt ctgatactta tccaaattac 180

aataaaaagt tcaagggcaa ggccacattg actgtagtcg tatcctccag cacagcctac 240

atgcagctca gcagcctgac atctgacgat tctgcggtct attactgtgc aagaaacggt 300

gattccgatt attactctgg tatggactac tggggtcaag gaacctcagt caccgtctcc 360

tca 363

<210>28

<211>121

<212>PRT

＜213〉Mustella

<220>

＜223〉mouse 2B6 variable region of heavy chain

<400>28

Gln Val Gln Leu Gln Gln Pro Val Thr Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Met Leu Ser Cys Lys Ala Ser Asp Tyr Pro Phe Thr Asn Tyr

20 25 30

Trp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asp Pro Ser Asp Thr Tyr Pro Asn Tyr Asn Lys Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Val Val Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Gly Asp Ser Asp Tyr Tyr Ser Gly Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210>29

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉3H7 variable region of heavy chain-CDR1

<400>29

Asp Ala Trp Met Asp

1 5

<210>30

<211>19

<212>PRT

＜213〉artificial sequence

<220>

＜223〉3H7 variable region of heavy chain-CDR2

<400>30

Glu Ile Arg Asn Lys Ala Asn Asn Leu Ala Thr Tyr Tyr Ala Glu Ser

1 5 10 15

Val Lys Gly

<210>31

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉3H7 variable region of heavy chain-CDR3

<400>31

Tyr Ser Pro Phe Ala Tyr

1 5

<210>32

<211>30

<212>PRT

＜213〉artificial sequence

<220>

＜223〉3H7 variable region of heavy chain-FWR1

<400>32

Glu Val Lys Phe Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

20 25 30

<210>33

<211>14

<212>PRT

＜213〉artificial sequence

<220>

＜223〉3H7 variable region of heavy chain-FWR2

<400>33

Trp Val Arg Gln Gly Pro Glu Lys Gly Leu Glu Trp Val Ala

1 5 10

<210>34

<211>30

<212>PRT

＜213〉artificial sequence

<220>

＜223〉3H7 variable region of heavy chain-FWR3

<400>34

Arg Phe Thr Ile Pro Arg Asp Asp Ser Lys Ser Ser Val Tyr Leu His

1 5 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Ile Tyr Tyr Cys

20 25 30

<210>35

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉3H7 variable region of heavy chain-FWR4

<400>35

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

1 5 10

<210>36

<211>345

<212>DNA

＜213〉Mustella

<220>

＜223〉mouse 3H7 variable region of heavy chain

<400>36

gaagtgaagt ttgaggagtc tggaggaggc ttggtgcaac ctggaggatc catgaaactc 60

tcttgtgctg cctctggatt cacttttagt gacgcctgga tggactgggt ccgccagggt 120

ccagagaagg ggcttgagtg ggttgctgaa attagaaaca aagctaataa tcttgcaaca 180

tactatgctg agtctgtgaa agggaggttc accatcccaa gagatgattc caaaagtagt 240

gtctacctgc acatgaacag cttaagagct gaagacactg gcatttatta ctgttatagt 300

ccctttgctt actggggcca agggactctg gtcactgtct ctgca 345

<210>37

<211>115

<212>PRT

＜213〉Mustella

<220>

＜223〉mouse 3H7 variable region of heavy chain

<400>37

Glu Val Lys Phe Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala

20 25 30

Trp Met Asp Trp Val Arg Gln Gly Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Glu Ile Arg Asn lys Ala Asn Asn Leu Ala Thr Tyr Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Pro Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu His Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Ile Tyr

85 90 95

Tyr Cys Tyr Ser Pro Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ala

115

<210>38

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉3H7 variable region of light chain-CDR1

<400>38

Arg Ala Ser Gln Glu Ile Ser Gly Tyr Leu Ser

1 5 10

<210>39

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉3H7 variable region of light chain-CDR2

<400>39

Ala Ala Ser Thr Leu Asp Ser

1 5

<210>40

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉3H7 variable region of light chain-CDR3

<400>40

Leu Gln Tyr Val Ser Tyr Pro Tyr Thr

1 5

<210>41

<211>23

<212>PRT

＜213〉artificial sequence

<220>

＜223〉3H7 variable region of light chain-FWR1

<400>41

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Thr Cys

20

<210>42

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉3H7 variable region of light chain-FWR2

<400>42

Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Arg Arg Leu Ile Tyr

1 5 10 15

<210>43

<211>32

<212>PRT

＜213〉artificial sequence

<220>

＜223〉3H7 variable region of light chain-FWR3

<400>43

Gly Val Pro Lys Arg Phe Ser Gly Ser Trp Ser Gly Ser Asp Tyr Ser

1 5 10 15

Leu Thr Ile Ser Ser Leu Glu Ser Glu Asp Phe Ala Asp Tyr Tyr Cys

20 25 30

<210>44

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉3H7 variable region of light chain-FWR4

<400>44

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

1 5 10

<210>45

<211>321

<212>DNA

＜213〉Mustella

<220>

＜223〉mouse 3H7 variable region of light chain

<400>45

gacatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60

ctcacttgtc gggcaagtca ggaaattagt ggttacttaa gctggcttca gcagaaacca 120

gatggaacta ttagacgcct gatctacgcc gcatccactt tagattctgg tgtcccaaaa 180

aggttcagtg gcagttggtc tgggtcagat tattctctca ccatcagcag ccttgagtct 240

gaagattttg cagactatta ctgtctacaa tatgttagtt atccgtatac gttcggaggg 300

gggaccaagc tggaaataaa a 321

<210>46

<211>107

<212>PRT

＜213〉Mustella

<220>

＜223〉mouse 3H7 variable region of light chain

<400>46

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Arg Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly

50 55 60

Ser Trp Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Val Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210>47

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer-SJ15R

<400>47

ggtcactgtc actggctcag gg 22

<210>48

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer-SJ16R

<400>48

aggcggatcc aggggccagt ggatagac 28

<210>49

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer-SJ17R

<400>49

gcacacgact gaggcacctc cagatg 26

<210>50

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer-SJ18R

<400>50

cggcggatcc gatggataca gttggtgcag catc 34

<210>51

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉fusion rotein-partial sequence

<400>51

Lys Lys Phe Ser Arg Ser Asp Pro Asn

1 5

<210>52

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉fusion rotein-partial sequence

<400>52

Gln Lys Phe Ser Arg Leu Asp Pro Asn

1 5

<210>53

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉fusion rotein-partial sequence

<400>53

Gln Lys Phe Ser Arg Leu Asp Pro Thr

1 5

<210>54

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉fusion rotein-partial sequence

<400>54

Lys Lys Phe Ser Arg Leu Asp Pro Thr

1 5

<210>55

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉fusion rotein-partial sequence

<400>55

Gln Lys Phe Ser His Leu Asp Pro Thr

1 5

<210>56

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉fusion rotein-partial sequence

<400>56

Lys Lys Phe Ser His Leu Asp Pro Thr

1 5

<210>57

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉fusion rotein-partial sequence

<400>57

Ala Pro Ser Ser Ser

1 5

<210>58

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉fusion rotein-partial sequence

<400>58

Val Pro Ser Met Gly Ser Ser Ser

1 5

1

The PCT/RO/134 table

PCT/RO/134 shows Chinese translation about the microbial preservation explanation

WO 2005/110474 PCT/US2005/016260

-176.1

Microorganism to specification sheets the _ _ _ _ page or leaf, the _ _ _ _ explanation of the described microorganism of row
		A. record in additional page about other further preservation information of explanation of preservation
Depositary institution's title: U.S. typical case thing preservation center
		Depositary institution address (comprising postcode and country): Virginia, USA, 20110-2209
Preservation date on August 13rd, 2002	Deposit number PTA-4591
Preservation date on August 13rd, 2002	Deposit number PTA-4591	B. have supplementary page in supplementary notes (in case of necessity) this column

		C. this explanation is done (if explanation is not done for all designated states) for following designated state
D. remark additionally (in case of necessity)
D. remark additionally (in case of necessity)		Following explanation will be submitted (writing out the classification of explanation, for example " numbering of preservation ") to international office subsequently

		E. this page or leaf of receives together with international application that (being filled in by the office of accepting) (authorize official) international office receives this page date (authorizing official)

Form PCT/RO/134

-176.2

The PCT application number:

The PCT/RO/134 table

U.S. typical case thing preservation center

Virginia, USA, 20110-2209

Preserving number preservation day

PTA-4592 on August 2nd, 2002

PTA-5958 on May 7th, 2004

PTA-5959 on May 7th, 2004

PTA-5960 on May 7th, 2004

PTA-5961 on May 7th, 2004

PTA-5962 on May 7th, 2004

PTA-5963 on May 7th, 2004

PTA-5964 on May 7th, 2004

Claims

1. humanized antibody, this antibody has the aminoacid sequence of the CDR that comprises a kind of monoclonal antibody, this monoclonal antibody combines with the cell foreign lands specificity of natural people Fc γ RIIB, and described bonded avidity is greater than zone, the extracellular bonded avidity of described antibody and natural human Fc gamma RIIA.

2. the described humanized antibody of claim 1, wherein said antibody comprises the CDR from monoclonal antibody 2B6 or 3H7.

3. the described humanized antibody of claim 1, wherein said antibody comprises the CDR of monoclonal antibody 1D5,2E1,2H9,2D11 or 1F2.

4. the described humanized antibody of claim 1, wherein said antibody comprises the humanization variable region with heavy chain district and light chain district, described heavy chain district has aminoacid sequence SEQ ID NO.24 or SEQ ID NO.37, and described light chain district has aminoacid sequence SEQ ID NO.18, SEQ ID NO.20, SEQ IDNO.22 or SEQ ID NO.46.

5. the described humanized antibody of claim 1, wherein said antibody comprises: have aminoacid sequence SEQ IDNO.1 or SEQ ID NO.29 VH CDR1, have the VH CDR2 of aminoacid sequence sequence SEQ ID NO.2 or SEQ ID NO.30 and have SEQ ID NO.3 or the VH CDR3 of SEQ ID NO.31 aminoacid sequence.

6. the described humanized antibody of claim 1, wherein said antibody comprises: the VL CDR1 with aminoacid sequence SEQ IDNO.8 or SEQ ID NO.38, have the VL CDR2 of aminoacid sequence SEQ ID NO.9, SEQID NO.10, SEQ ID NO.11 or SEQ ID NO.39 and have aminoacid sequence SEQ ID NO.12 or the VL CDR3 of SEQ ID NO.40.

7. the described humanized antibody of claim 1 comprises the CDR district of variation, the CDR district of wherein said variation comprise among the VL CDR2 at least one is amino acid modified.

8. the described humanized antibody of claim 7, wherein said at least one amino acid modified 50 place, position that are included in are replaced by tyrosine.

9. the described humanized antibody of claim 7, wherein said at least one amino acid modified 51 place, position that are included in are replaced by L-Ala.

10. each described humanized antibody of claim 1-9, the framework region of wherein said antibody does not contain amino acid modified.

11. the fragment of the described antibody of claim 1, wherein said fragment are F (ab ') 2 fragments or F (ab) fragment.

12. the described antibody of claim 1, wherein said antibody is single-chain antibody.

13. the described antibody of claim 1, wherein said antibody operationally is connected with heterologous polypeptide.

14. the described antibody of claim 1, wherein said antibody combines with therapeutic agent.

15. the described antibody of claim 14, wherein said therapeutic agent is a cytotoxin.

16. the described antibody of claim 1, wherein said antibody blocking Ig-Fc combines with Fc γ RIIB's.

17. the described antibody of claim 1, wherein said antibody more effectively slows down tumor growth than Rituxin.

18. isolating nucleic acid, it comprises the nucleotide sequence of the coding described antibody of claim 1 or its segmental heavy chain or light chain.

19. carrier, it comprises the described nucleic acid molecule of claim 18.

20. carrier, it comprises first nucleic acid molecule of encoding heavy chain and second nucleic acid molecule of coding light chain, and described heavy chain and light chain are the described antibody of claim 1 or its segmental heavy chain and light chain.

21. the described carrier of claim 19, it is an expression vector.

22. host cell, it contains the described carrier of claim 19.

23. host cell, it contains first nucleic acid that is operably connected with allogeneic promoter and second nucleic acid that is operably connected with identical or different allogeneic promoter, described first nucleic acid and second nucleic acid encode the respectively heavy chain and the light chain of the described antibody of claim 1.

24. treatment is the method for cancer of feature with cancer antigen in the patient, this method comprises each the described humanized antibody of claim 1-9 to described patient's administering therapeutic significant quantity, and combines with described cancer antigen-specific and have Cytotoxic antibody.

25. the described method of claim 24, wherein said cancer are mammary cancer, ovarian cancer, prostate cancer, cervical cancer or carcinoma of the pancreas.

26. the described method of claim 24, wherein said cytotoxic antibody are trastuzumab (trastuzumab), Mabthera (rituximab), IC14, Edrecolomab (edrecolomab), Cetuximab (cetuximab), VITAXIN ^TM, Campath 1H/LDP-03, epratuzumab (epratuzumab) or ZEVLIN ^TM

That 27. the described method of claim 24, wherein said cancer antigen are MAGE-I, MAGE-3, BAGE, GAGE-1, GAGE-2, N-acetylglucosaminyltransferase, p15, beta-catenin is white (β-carenin), MUM-1, CDK4, HER-2/neu, human papillomavirus-E6, human papillomavirus-E7 or MUC-1.

28. the described method of claim 24, wherein said cancer antigen are breast cancer antigen, ovarian cancer antigen, prostate cancer antigen, cervical cancer antigen or carcinoma of the pancreas antigen.

29. also comprising, the described method of claim 24, this method give one or more other cancer therapy.

30. the described method of claim 29, wherein said other cancer therapy is selected from: chemotherapy, immunotherapy, radiotherapy, hormonotherapy or surgical operation.

31. the described method of claim 24, wherein said patient is the people.

32. pharmaceutical composition, it contains: i) each described humanized antibody of claim 1-9 of treatment significant quantity; Ii) with cancer antigen-specific bonded cytotoxic antibody; Iii) pharmaceutically acceptable carrier.

33. the method for treatment autoimmune disorder in the patient, described method comprises each described humanized antibody to the claim 1-9 of described patient's administering therapeutic significant quantity.

34. the described method of claim 33, wherein said autoimmune disorder are rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Rieter ' s syndrome, psoriatic or lupus erythematosus.

35. the described method of claim 33, this method also comprise one or more anti-inflammatory agenies to described patient's administering therapeutic significant quantity.

36. the described method of claim 33, this method also comprise one or more immunomodulators to described patient's administering therapeutic significant quantity.

37. the described method of claim 33, wherein at least a immunomodulator is an organic molecule.

38. the described method of claim 37, wherein said organic molecule are methotrexate, leflunomide (leflunomide), endoxan, cyclosporine A, FK506, mycophenolate mofetil (mycophenolatemofetil), Wyeth-Ayerst Laboratories, mizoribine (mizoribine), Gusperimus (deoxyspergualin), brequinar (brequinar), malonitrolamide, steroid or reflunomide.

39. the described method of claim 35, wherein at least a anti-inflammatory agent is the non-steroid antiphlogiston.

40. the described method of claim 39, wherein said on-steroidal AID are acetylsalicylic acid, Ibuprofen BP/EP, diclofenac, nabumetone (nabumetone), Naproxen Base (naproxen) or Ketoprofen (ketoproten).

41. comprising to described patient, the method for the anaphylactic disease of treatment or prevention IgE mediation in this patient who needs is arranged, this method treat each described humanized antibody among the claim 1-9 of significant quantity.

42. the described method of claim 41, the anaphylactic disease of wherein said IgE mediation is asthma, allergic rhinitis, gi tract allergy, hypereosinophilic syndrome, conjunctivitis, or glomerulonephritis.

43. improve the method for antibody-mediated cytotoxicity in the patient who accepted the cytotoxicity Antybody therapy, described method comprises to described patient is enough to strengthen each described humanized antibody among the claim 1-9 of amount of cytotoxicity of described cytotoxic antibody.

44. the method for diagnosis intrasubject immunological disease, this method comprises:

(a) will contact with each described humanized antibody of claim 1-9 of significant quantity from the biological sample of described individuality; With

(b) combination of the described antibody of detection,

Wherein detecting be higher than background or standard level described detects mark and shows that described individuality suffers from autoimmune disorder.

45. the described method of claim 44, the wherein said mark that detects is chemiluminescent labeling, enzyme labelling, fluorescent mark or radio-labeling.

46. strengthen individual immunoreactive method to vaccine composition, described method comprises to described individuality and gives claim 1-9 each described humanized antibody and vaccine composition, and described humanized antibody gives the immunoreactive significant quantity of described vaccine composition to strengthen described individuality.

47. it is patient's the method for the cancer of feature that treatment suffers from cancer antigen, described method comprises each described humanized antibody to the claim 1-9 of described patient's administering therapeutic significant quantity, and the antibody of wherein being used reduces the number of the cancer cells of expressing Fc γ RIIB.

48. it is patient's the method for the cancer of feature that treatment suffers from cancer antigen, described method comprises each described humanized antibody to the claim 1-9 of described patient's administering therapeutic significant quantity, wherein the cleaning antibody of being used express the cancer cells of Fc γ RIIB.