CN101218351A - Anti-CD19 antibodies and uses in oncology - Google Patents

Abstract

The invention relates to immunotherapeutic compositions and methods for the treatment of B cell diseases and disorders in human subjects, such as, but not limited to, B cell malignancies, using therapeutic antibodies that bind to the human CD 19 antigen and that preferably mediate human ADCC. The present invention relates to pharmaceutical compositions comprising human or humanized anti-CD 19 antibodies of the IgGl or IgG3 human isotype. The present invention relates to pharmaceutical compositions comprising human or humanized anti-CD 19 antibodies of the IgG2 or IgG4 human isotype that preferably mediate human ADCC. The present invention also relates to pharmaceutical compositions comprising chimerized anti-CD19 antibodies of the IgGl, IgG2, IgG3, or IgG4 isotype that mediate human ADCC. In preferred embodiments, the present invention relates to pharmaceutical compositions comprising monoclonal human, humanized, or chimeric anti-CD 19 antibodies.

Description

Anti-CD 19 antibodies and the application in oncology thereof

The present invention partly be the fund of the National Cancer Institute of U.S.'s National Institutes of Health (National Institutes ofHealth) (National Cancer Institute) number for federal government's fund assistance of CA81776, CA105001 and CA96547 under and the fund of the state-run allergy of U.S.'s National Institutes of Health (National Institutes of Health) and infectious disease research institute (National Institute of Allergy and Infectious Disease) number finish under federal government's fund assistance of AI56363.United States Government enjoys certain right of the present invention.

The application requires the U.S. Provisional Patent Application 60/653 of application on February 15th, 2005 according to United States Code the 35th the 119th (e) money of volume (35U.S.C. § 119 (e)), the U.S. Provisional Patent Application 60/702 of application on July 22nd, 587 and 2005,063 right of priority, and it is incorporated herein by reference with its integral body.

1. brief introduction

The present invention relates in the experimenter, use the method for carrying out B cell disorder or disease treatment, comprise the B cell tumour with people CD19 antigen bonded therapeutic antibodies.In preferred embodiments, the cytotoxicity (ADCC) of the preferred Mediated Human antibody dependent cellular mediation of the therapeutic anti-CD 19 antibodies of the compositions and methods of the invention.The invention further relates to the composition of the anti-CD 19 antibodies that comprises the IgG1 people, humanized or chimeric and/or IgG3 people's isotype.The invention further relates to and comprise the IgG2 people, humanized or chimeric and/or IgG4 people's isotype, the composition of the anti-CD 19 antibodies of the ADCC of preferred Mediated Human.The present invention also comprises anti-CD 19 antibodies monoclonal people, humanized or chimeric.

2. background of the present invention

The B cell surface marker has been thought the target of B cell disorder or disease, autoimmune disease and transplant rejection at large.The example of B cell surface marker comprises CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85 and CD86 leukocyte surface mark.Developed the antibody with these mark particular combination, and some antibody disease and disorderly treatment have been used for by test.

For example, verified, at the therapy based on monoclonal antibody chimeric or labelled with radioisotope (mAb) of the CD20 cell surface molecule of the pernicious counterpart that is specific to mature B cell and they (people such as Tedder, the Immunol.Today 15:450-454 (1994) of therapy in effective body of non-hodgkin's lymphomas; People such as Press, Hematology, 221-240 (2001); People such as Kaminski, N.Engl.J.Med, 329:459-465 (1993); Weiner, Semin.Oncol, 26:43-51 (1999); People such as Onrust, Drugs, 58:79-88 (1999); People such as McLaughlin, Oncology, 12:1763-1769 (1998); People such as Reff, Blood, 83:435-445 (1994); People such as Maloney, Blood, 90:2188-2195 (1997); People such as Maloney, J.Clin.Oncol, 15:3266-3274 (1997); People such as Anderson, Biochem.Soc.Transac, 25:705-708 (1997)).Find that also the anti-CD-20 monoclonal antibody therapy can improve arthritis deformans, systemic lupus erythematosus, idiopathic thrombocytopenic purpura and hemolytic anemia, and the clinical manifestation of other immune-mediated disease (people such as Silverman, Arthritis Rheum.48:1484-1492 (2002); People such as Edwards, Rheumatology, 40:1-7 (2001); People such as De Vita, Arthritis Rheumatism, 46:2029-2033 (2002); People such as Leandro, Ann.Rheum.Dis.61:883-888 (2002); People such as Leandro, Arthritis Rheum.46:2673-2677 (2001)).Anti-CD22 monoclonal antibody LL-2 is proved to be effectively (Goldenberg U.S. Patent number: 6,134,982 and 6,306,393) in treatment stands the malignant lymphoma patient of the aggressiveness of chemotherapy and recurrent.Will resist CD20 (IgG1) antibody, RITUXAN ^TMBe successfully used to some treatment of diseases, for example be grown up immune thrombopenic purpura, rheumatic arthritis and autoimmune hemolytic anemia (people such as Cured, WO 00/67796).No matter the validity of this therapy, according to the CD20 immunotherapy, most acute lymphoblastic leukemia (ALL) and many other B cell malignancies are not expressed CD20, express CD20 on low-level or lost CD20 expression (people such as Smith, Oncogene, 22:7359-7368 (2003)).In addition, the expression of CD20 is not the omen of antagonism CD20 therapy, only has the Fei Huoqijinshi malignant lymphoma patient of half that the immunotherapy of CD20 control is reacted.

People's CD19 molecule is a clearly cell surface receptor of the structure expressed on the surface of people's B cell, described B cell includes but are not limited to, and the B cell of pre B cell (pre-B cell), early development (that is: jejune B cell), sophisticated B cell are by end differentiation becoming plasmocyte and Malignant B cell.Before most-B acute lymphoblastic leukemia (ALL), the Fei Huoqijinshi malignant lymphoma, B cell lymphocytic leukemia (CLL), PL, hairy cell leukemia, common acute Lymphocytic leukemia and some non-acute lymphoblastic leukemias are all expressed CD19 (people such as Nadler, J.Immunol.131:244-250 (1983), people such as Loken, Blood, 70:1316-1324 (1987), people such as Uckun, Blood, 71:13-29 (1988), people such as Anderson, 1984.Blood, 63:1424-1433 (1984), Scheuermann, Leuk.Lymphoma, 18:385-397 (1995)).Further specify it in the expression of the CD19 on the plasmocyte and can for example express (people such as Grossbard, Br.J.Haematol, 102:509-15 (1998) on multiple myeloma, plasmoma, the Fahrenheit knurl in different B glucagonomas; People such as Treon, Semin.Oncol, 30:248-52 (2003)).Be different from CD20, when combining with anti-CD 19 antibodies, CD19 antigen is considered at high level expression, and is integrated in the cell.

CD19 antigen also be many suggestions be used for one of target of immunotherapy.But, recognize owing to the cell internalizing effect causes and can not utilize as target, be considered to cause obstruction for the exploitation of the treatment plan that can be successfully used to human subjects.CLB-CD19 antibody (the IgG2a mAb of anti-CD19 mouse) is proved to be the growth that suppressed to implant the human tumor in athymic mouse people such as (, Cancer Research, 55:840-846 (1995)) Hooijberg.In another research, end user's IgG1 Fc fragment is come the murine antibody FMC63 (IgG2a) of chimeric monoclonal.Be somebody's turn to do administration for the chimeric antibody of the SCID mouse with human B cell malignant lymphoma (heteroplastic transplantation model), do not induce the cytotoxicity of complement-mediated or the cytotoxicity (ADCC) of antibody dependent cellular mediation, but cause obviously the killing and wounding of the tumour cell transplanted (people such as Geoffrey, Cancer Immunol.Immunother.41:53-60 (1995)).

The result that the xenotransplantation mouse model of use tumour transplatation is obtained has caused using the research of the anti-CD 19 antibodies of mouse in human patients.The CLB-CD19 antibody of mouse is diagnosed as gradual Fei Huoqijinshi malignant lymphoma, and six patients that use above-mentioned traditional remedies (chemotherapy or radiotherapy) to fail.Give these patients from 225 to 1, total antibody dosage of 000mg (people such as Hekman, Cancer Immunol.Immunotherapy, 32:364-372 (1991)).Though the circulating tumor cell latter two patient who injects antibody has temporarily reduced, only there is a patient to realize partly alleviating after two cycles at Antybody therapy.From the patient of this group's refractory, can not draw conclusion about therapeutic efficacy.

Afterwards, these investigators have shown that the antitumous effect of uncombined CD20mAb in transplantation model is than the much better (people such as Hooijberg of the antitumous effect of CD19mAb, people such as Cancer Res.55:840-846 (1995) and Hooijberg, Cancer Res.55:2627-2634 (1995)).In addition, when uniting when using CD19 and CD20mAb, they do not observe for the accumulation of tumor incidence or synergistic effect people such as (, Cancer Res.55:840-846 (1995)) Hooijberg.Though heteroplastic animal model is considered to the bad prediction indicator of effect among the experimenter, the negativity result who obtains in these animals has stoped people for the interest with the therapy of naked anti-CD 19 antibodies.

Utilization based on the immunotoxin of anti-CD 19 antibodies has produced disappointed result equally.In the clinical trial in the early stage, (mouse IgG1 mAb) is attached on the plant poison ricin with the B4 anti-CD 19 antibodies, and the multiple myeloma human patients (people such as Grossbard who uses above-mentioned traditional remedies to fail, British Journal of Haematology, 102:509-515 (1998)), high Fei Huoqijinshi malignant lymphoma (people such as Grossbard, Clinical CancerResearch, 5:2392-2398 (1999)) and the B cell malignancies of refractory (people such as Grossbard, Blood, 79:576-585 (1992)).These tests have proved that usually the B4-ricin cooperates the security that gives the mankind; But, and use RITUXAN ^TMClinical trial compare confusion, and speed of response as a result disappointing (people such as Grossbard, Clinical CancerResearch, 5:2392-2398 (1999)).In addition, obviously human antimouse antibody (HAMA) reaction or the anti-ricin antibody of people (HARA) reaction have taken place in some patient.

In another experiment, combine seven slight Fei Huoqijinshi malignant lymphoma patients treating with traditional remedies before treating (people such as Vlasveld with the low dose of interleukin II of the CLB-CD19 antibody of mouse and injection continuously, Cancer Immunol.Immunotherapy, 40:37-47 (1995)).Part in a leukaemic, occurs and alleviate, and observe circulation B cell greater than 50% reduction.In 4 in 5 residue patients that measure, the quantity of circulation B cell does not change.Therefore, mouse-anti CD19 antibody and in human body, produced to be used for that effect estimates unmatchful according to data based on the therapeutic evaluation of the immunotoxin of anti-CD18 antibody.

3. summary of the invention

The present invention relates to be used for use the therapeutic antibodies that combines and preferably mediate ADCC with people CD19 antigen to treat B cell disease and disorder, for example, but be not limited to the immunotherapeutic composition of B cell malignancies and method the experimenter.The present invention relates to comprise the medicine of the anti-CD 19 antibodies people, humanized IgG1 and/or IgG3 people's isotype.The present invention relates to comprise the people, humanized IgG2 and/or IgG4 people's isotype, the ADCC anti-CD 19 antibodies of preferred Mediated Human.The present invention relates to comprise the pharmaceutical composition of anti-CD 19 antibodies of IgG1, IgG2, IgG3 or the IgG4 isotype of chimeric Mediated Human ADCC.In preferred embodiments, the present invention relates to comprise the pharmaceutical composition of anti-CD 19 antibodies monoclonal people, humanized or chimeric.

Described to be used for the treatment of to be diagnosed as deriving from B cell or its precursor, included but not limited to the experimenter's of acute lymphoblastic leukemia (ALL), hodgkin's malignant lymphoma, Fei Huoqijinshi malignant lymphoma, B cell lymphocytic leukemia (CLL), multiple myeloma, follicular lymphoma, amphicyte malignant lymphoma, prolymphocyte leukemia, hairy cell leukemia, common acute Lymphocytic leukemia and the leukemic B cell malignancies of some non-acute lymphocytoblasts therapeutic composition and mode.

Use the immunotherapy at CD19 of transgene mouse model evaluation in the experimenter to illustrate method of the present invention.

In one embodiment, the invention provides a kind of pharmaceutical composition that is included in the monoclonal people's or humanized IgG1 or IgG3 people's isotype the anti-CD 19 antibodies in the pharmaceutically acceptable carrier.In another embodiment, the invention provides a kind of pharmaceutical composition that is included in the anti-CD 19 antibodies that comprises the monoclonal chimeric IgG1 that treats significant quantity or IgG3 people's isotype in the pharmaceutically acceptable carrier.In relevant embodiment, the significant quantity in the treatment of the anti-CD 19 antibodies of monoclonal chimeric IgG1 or IgG3 people's isotype is to be less than every kg weight in patients 1mg.In other related embodiment, the significant quantity in the treatment of the anti-CD 19 antibodies of monoclonal chimeric IgG1 or IgG3 people's isotype is greater than every kg weight in patients 2mg.

According to an aspect, the invention provides a kind of pharmaceutical composition of the monoclonal people's or humanized anti-CD 19 antibodies treatment significant quantity of the cytotoxicity (ADCC) that is included in the mediation of Mediated Human antibody dependent cellular in the pharmaceutically acceptable carrier.According to another aspect, the invention provides the pharmaceutical composition of the active monoclonal chimeric anti-CD 19 antibodies of a kind of cytotoxicity (ADCC) that is included in the mediation of Mediated Human antibody dependent cellular in the pharmaceutically acceptable carrier and/or CDC (CDC) and/or apoptosis.

The present invention relates to a kind of in human body the method for treatment B cell malignancies, comprise the people's or humanized IgG1 or IgG3 people's isotype the anti-CD 19 antibodies of the human monoclonal that needs, its quantity enough consumes round-robin B cell.The invention still further relates to a kind of method of in human body, treating the B cell malignancies, comprise the anti-CD 19 antibodies of the people's or the mediation of humanized Mediated Human antibody dependent cellular the cytotoxicity (ADCC) of the human monoclonal that needs, its quantity enough consumes round-robin B cell.The present invention relates to a kind of in human body the method for treatment B cell malignancies, comprise the monoclonal people's or humanized IgG1 or IgG3 people's isotype the anti-CD 19 antibodies of the human patients of the such treatment of needs being treated significant quantity.

In one embodiment, the invention provides a kind of in human patients the method for treatment B cell malignancies, comprise the anti-CD 19 antibodies of the human patients of the such treatment of needs being treated the monoclonal people's or the mediation of humanized Mediated Human antibody dependent cellular the cytotoxicity (ADCC) of significant quantity.In another embodiment, the invention provides the method for the early stage disease that a kind of treatment causes by the B cell malignancies in human patients, comprise the monoclonal anti-CD 19 antibodies of cytotoxicity (ADCC) that people to the such treatment of needs treats the Mediated Human antibody dependent cellular mediation of significant quantity.In further embodiment, the invention provides a kind of method of in human patients, treating the B cell malignancies, comprise the anti-CD 19 antibodies of cytotoxicity (ADCC) of the human subjects of the such treatment of needs being treated the monoclonal Mediated Human antibody dependent cellular mediation of significant quantity, wherein the experimenter was not subjected to treating malignant tumor in the past.Another embodiment of the present invention provides a kind of method for the treatment of the B cell malignancies in human patients, comprise the monoclonal anti-CD 19 antibodies of cytotoxicity (ADCC) of the human patients of the such treatment of needs being treated the Mediated Human antibody dependent cellular mediation of significant quantity, wherein the B cell malignancies is the CD19 male.In a further embodiment, the invention provides a kind of method of in human patients, treating the B cell malignancies, comprise the monoclonal anti-CD 19 antibodies of cytotoxicity (ADCC) of the human patients of the such treatment of needs being treated the Mediated Human antibody dependent cellular mediation of significant quantity, wherein contain at least 1 monocyte number in the blood circulation of the every dL of human patients.

3.1 definition

As used herein, term " antibody " and " antibody " (immunoglobulin (Ig)) refer to monoclonal antibody (monoclonal antibody that comprises total length), polyclonal antibody, the multi-specificity antibody that forms by at least two complete antibody (for example: bi-specific antibody), people's antibody, human antibody, camelised antibody, chimeric antibody, strand Fvs (scFv), single-chain antibody, single domain antibody, domain antibodies, Fab fragment, F (ab ') ₂Fragment, performance require bioactive antibody fragment, the Fvs (sdFv) of disulphide connection and the antigenic determinant binding fragment of anti-Idiotype (anti-Id) antibody (comprising, for example: for the anti-Id antibody of antibody of the present invention), intracellular antibody and above all antibody.Especially, antibody comprises the immunocompetence fragment of immunoglobulin molecules and immunoglobulin molecules, comprises the molecule of antigen binding site that is:.Immunoglobulin molecules can have arbitrary type (for example: IgG, IgE, IgM, IgD, IgA and IgY), class (for example: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

The different four dimerization glycoprotein that normally about 150,000 dalton of natural antibody are made up of two identical light (L) chains and two identical weights (H) chain.Each light chain is connected on the heavy chain by the disulfide linkage of a covalency, and changes the number of disulfide linkage between the heavy chain of different immunoglobulin (Ig) isotypes.Each weight and light chain also have the interior disulphide bridges of chain at regular interval.Each heavy chain has variable region (VH) at the one end, is thereafter some constant regions.Each light chain has variable region (VL) at the one end, has constant region at its other end; The constant region of light chain links to each other with first constant region of heavy chain, and variable region of light chain links to each other with variable region of heavy chain.Specified amino acid residues is considered to form linker between the variable region of light chain and heavy chain.Such antibody can derive from arbitrary Mammals, includes but not limited to people, monkey, pig, horse, rabbit, dog, cat, mouse etc.

Some part that term " variable " refers to variable region in antibody different widely fact on sequence, and cause each specific antibody for its specific antigenic binding specificity.But mutability is not equally distributed in the variable region of antibody.It concentrates in the fragment that is called complementarity-determining region (CDRs) of variable region of light chain and variable region of heavy chain.The height constant of variable region partly is known as skeleton district (FR).The variable region of each natural heavy chain and light chain comprises four FR districts, mainly takes the beta sheet structure, links to each other by three CDRs that form the ring connection, and becomes the part of beta sheet structure sometimes.CDRs in each chain closely combines by the CDRs of FR district and another chain is approximate, the antigen haptophoric formation that helps antibody is (referring to people such as Kabat, Sequences of Proteins of Immunological Interest, 5thEd.Public Health Service, National Institutes of Health, Bethesda, MD (1991)).Constant region directly is not included in the bonded antigen usually, but may influence antigen bonded avidity, and may demonstrate various effector functions, for example participates in the antibody among the ADCC.

What term as used herein " hypervariable region " referred to antibody causes being attached to amino-acid residue on its antigen.The hypervariable region from " complementarity-determining region " or " CDR " (for example: at the residue 24-34 of variable region of light chain (L1), 50-56 (L2) and 89-97 (L3) with at the residue 31-35 of variable region of heavy chain (H1), 50-65 (H2) and 95-102 (H3) comprises; People such as Kabat, Sequences of Proteins of Immunological Interest, 5th Ed.Public HealthService, National Institutes of Health, Bethesda, MD (1991)) and/or (for example: at the residue 26-32 of variable region of light chain (L1), 50-52 (L2) and 91-96 (L3) with at the residue 26-32 of variable region of heavy chain (H1), 53-55 (H2) and 96-101 (H3) from those residues of " hypermutation ring "; Chothia and Lesk, J.Mol Biol, 196:901-917 (1987))." skeleton structure " or " FR " residue is except those variable region residues the hypervariable region residue as defined here, and comprises chimeric, humanized, the people, domain antibodies, binary molecule (diabodies), vaccine body (vaccibodies), linear antibody and bi-specific antibody.

Term " monoclonal antibody " refers to the antibody that obtains from one group of similar basically antibody as used herein, that is: except comprising the independent antibody of similar number with the naturally occurring sudden change of the potential that exists more on a small quantity.Monoclonal antibody is a high degree of specificity, and it is corresponding with single antigen site.In addition, opposite with tradition (polyclone) antibody preparation that generally comprises with the corresponding different antibodies of different determinants (antigenic determinant), each monoclonal antibody be with antigen on single determinant corresponding.Except its specificity, monoclonal antibody is not favourable by the aspect of other immunoglobulin (Ig) production cell contamination synthetic by hybridoma.In addition, monoclonal antibody can by with the coding heavy chain of monoclonal antibody and light chain gene stably or the cell of transient transfection produce.

Modifier " monoclonal " shows the characteristic of the antibody that obtains from the colony of the homogeneous basically of antibody, and is not considered to the antibody technique of needs by any special method.Term as used herein " monoclonal " refers to derive from the clonal population of cell, comprises the clone's of any eucaryon, protokaryon or phage antibody, rather than the method for engineered antibody.For example, can prepare by hybridoma method according to monoclonal antibody used in the present invention, described method is at first by people such as Kohler, Nature, 256:495 (1975), maybe can prepare by any recombinant DNA method (referring to: for example, U.S. the patent No.: 4,816,567), it comprises use for example people such as Clackson, Nature, method described in the people such as 352:624-628 (1991) and Marks, J.Mol.Biol.222:581-597 (1991) is separated from phage antibody library.These methods can be used for producing monoclonal Mammals, chimeric, humanized, the people, domain antibodies, binary molecule (diabodies), vaccine body (vaccibodies), linear antibody and bi-specific antibody.

The part that term " chimeric " antibody comprises wherein heavy chain at least and/or light chain with derive from particular types belong to the class of specific antibodies or the antibody of subclass in the identical or homology of corresponding sequence, and at least one of this chain other part with derive from another kind belong to the class of another antibody or the antibody of subclass in the identical or homology of corresponding sequence, and such antibody fragment, as long as it shows the biological activity (U.S. Patent number of wanting: 4,816,567; People such as Morrison, Proc.Natl.Acad.ScL USA, 81:6851-6855 (1984)).The chimeric antibody that this place is concerned about comprises " primateization (primatized) " antibody, described " primateization (primatized) " antibody sources in non-human primate (for example: gerontogeous monkey, variable region antigen binding sequence for example: baboon, macaque or absorption and elution test macaque) and people's constant region sequence (U.S. Patent number: 5,693,780).

" people source " form of inhuman (for example: mouse) antibody is the chimeric antibody that comprises the minmal sequence that derives from inhuman immunoglobulin (Ig).For most of situation, humanized antibody is human normal immunoglobulin (antibody of acceptor), and the residue of hypervariable region that wherein comes autoreceptor is by for example mouse, rat, rabbit or the residue of hypervariable region with specificity, avidity and active non-human primate of requirement replace from inhuman species (antibody of donor).In some cases, skeleton district (FR) residue of human normal immunoglobulin is replaced by corresponding inhuman residue.In addition, humanized antibody can be included in undiscovered residue in the antibody of the antibody of acceptor or donor.Carry out these modifications so that improve the performance of antibody further.In general, humanized antibody comprises at least one basically, and usually two, the variable region, wherein all or whole basically hypermutation rings corresponding to the hypermutation ring of non-human immunoglobulin, and all or whole basically FR be the FR of human normal immunoglobulin sequence.In certain embodiments, humanized antibody comprises the constant region for immunoglobulin (Fc) of at least a portion, generally is the constant region of human normal immunoglobulin.Relevant further details, referring to, people such as Jones, Nature, 321:522-525 (1986); People such as Riechmann, Nature, 332:323-329 (1988) and Presta, Curr.Op.Struct.Biol.2:593-596 (1992).

" people's antibody " can be derive from people's antibody or from " design " produce special corresponding to antigenic attack, and the antibody that obtains in can the genetically modified organism of people's antibody by any method preparation known in the art.According to preferable methods, people's the heavy chain and the element of light chain position are imported in the cell strain that derives from embryonic stem cell line, described clone comprises endogenous heavy chain and and the target cracking of light chain position.Genetically modified organism can be synthesized the people's antibody that is specific to the human antigen, and organism can be used for producing people's antibody-secreting hybridoma.People's antibody can also be the coded antibody of nucleotide sequence in heavy chain and light chain origin one or more sources of coming from people DNA.Can also be with method and the phage display technology or the external activating B cell of gene or karyomit(e) transfection, all these technology known in the art make up people's antibody completely.

" CD19 " antigen refers to the antigen of about 90kDa of being identified by HD237 or B4 antibody people such as (, Leukemia ResearchII, 12:1119 (1987)) Kiesel.CD19 is fixed on the cell, described cell is that the differentiation of cell is divided into plasmocyte by end from the stem cell stage by B, include but are not limited to pre B cell, B cell (comprising inmature B cell, antigenic stimulation B cell, memory B cell, plasmocyte and bone-marrow-derived lymphocyte) and folliculus branch cell.Also CD19 is fixed on people's the B cell of fetal tissue.In preferred embodiments, the target CD19 antigen of antibody of the present invention is people CD19 antigen.

The reaction of " cytotoxicity of antibody dependent cellular mediation " and the mediation of " ADCC " phalangeal cell, wherein nonspecific cytotoxin cell (for example: natural killer (NK) cell, neutrophilia neutrophil(e) cell and scavenger cell) is identified in bookbinding bonded antibody on the target cell, causes the dissolving of target cell afterwards.In preferred embodiments, such cell is people's a cell.Do not want to be limited to any specific mechanism of action mechanism of action, the cytotoxin cell of these mediations ADCC is expressed Fc acceptor (FcRs) usually.The primary cell of mediation ADCC, NK cell is expressed Fc γ RIII, and monocytes Fc γ RI, Fc γ RII, Fc γ RIII and/or Fc γ RIV.To on hematopoietic cell, express FcR and be summarized in Ravetch and Kinet, Annu.Rev.Immunol.9:457-92 (1991).In order to measure the ADCC activity of molecule, can carry out external ADCC test, for example U.S. Patent number: 5,500,362 or 5,821,337 is described.The useful effector cell of this test comprises peripheral blood monocyte (PBMC) and natural killer (NK) cell.Optionally, or additionally, can measure the ADCC activity of molecule (s) of interest in vivo, for example as people such as Clynes, PNAS (USA) is in the disclosed animal model of 95:652-656 (1998).

" cytotoxicity of complement-dependent " or " CDC " refer to initial complement activation of molecule and the ability of dissolving target under the situation that has complement to participate in.The complement activation approach originates from first combination of elements of complement system (CIq) to the molecule that is compounded with isogeneic (for example, antibody).In order to measure complement activation, can carry out the CDC test, for example, and as people such as Gazzano-Santaro, J.Immunol.Methods, 202:163 (1996) is described.

" effector cell " expresses one or more FcR, and carries out the white corpuscle of operon function.Preferably, cell expressing is Fc γ RI, Fc γ RII, Fc γ RIII and/or Fc γ RIV at least, and carries out ADCC operon function.The example of the human leukocyte of mediation ADCC comprises peripheral blood monocyte (PBMC), natural killer (NK) cell, monocyte, cytotoxic T cell and neutrophil(e) cell; Wherein preferred PBMC and NK cell.The effector cell is people's a cell in preferred embodiments.

Term " Fc acceptor " or " FcR " are used to describe the acceptor in the Fc district that is attached to antibody.Preferred FcR is the people FcR of native sequences.In addition, preferred FcR is in conjunction with IgG antibody (γ acceptor), and comprises Fc γ RI, Fc γ RII, Fc γ RIII and Fc γ RIV subclass, comprises the FcR of the substituting splicing form of allelic variant and these acceptors.Fc γ RII acceptor comprises Fc γ RIIA (a kind of " activated receptor ") and Fc γ RIIB (a kind of " inhibition acceptor "), and it has similar main at the different aminoacid sequence of its tenuigenin structural domain.Activated receptor Fc γ RIIA comprises the activation motif (ITAM) based on immunity receptor tyrosine in its tenuigenin structural domain.(referring to, Daeron, Annu.Rev.Immunol, 15:203-234 (1997)).At Ravetech and Kinet, Annu.Rev.Immunol, 9:457-92 (1991); People such as Capel, Immunomethods, 4:25-34 (1994); With people such as de Haas, J.Lab.Clin.Med, 126:330-41 has commented FcR in (1995).To identify the FcR of coming out after other FcR comprises, its term " FcR " from here comprises.This term also comprises newborn acceptor, FcRn, and it is responsible for IgG with mother and is delivered to fetus people such as (, Immunol, people such as 117:587 (1976) and Kim, J.Immunol, 24:249 (1994)) Guyer.

" Fv " is the minimum antibody fragment that comprises holoantigen identification and binding site.This zone by a heavy chain and variable region of light chain with closely, non-covalent or covalently bound dimer formed.It is in three interactional structures of CDR of each variable domains and forms the antigen haptophore on the dimeric surface of VH-VL.In general, six CDR give the antigen-binding specificity for antibody.But,, have lower avidity though compare with whole binding sites even one variable domains (or only comprising three for half of the Fv of the CDR of antigen-specific) has the ability of identification and conjugated antigen.

The antibody that is used for the treatment of described herein is term as known in the art for " avidity " of epitope, refers to that antibodies arrives the degree or the intensity of epitope.Avidity can be measured and/or represents with the many known methods in this area, includes but not limited to, balance ionization constant (KD or Kd), apparent equilibrium ionization constant (KD ' or Kd ') and IC ₅₀(causing that in competitive trials 50% suppresses needed amount).Certainly, for purposes of the invention, avidity is the average avidity for the antibody of giving determined number that is incorporated into epitope.The Ig milligram number according to every milliliter of serum of KD ' value representation of the mgIgG of every mL of this place report is though can use blood plasma.When the administration matrix that antibody affinity is used as methods of treatment described herein, or during as the candidate of methods of treatment described herein, can be before treatment and/or the period detecting antibody affinity, and whether the value of gained can be that suitable candidate is used to treatment by the clinical judgment human patients.

" epitope " is term known in the art, refers to show any chemical part that specificity is incorporated into antibody." epitope " also can comprise antigen, and it is part or the molecule that comprises epitope, and, similarly, also be incorporated into antibody specifically.

" B cell surface marker " used herein is the antigen of expressing on the B cell surface, described B cell can use with its bonded vehicle as target.The B cell surface marker of example comprises CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD37, CD53, CD72, CD73, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85 and CD86 leukocyte surface mark.Compare with mammiferous other non-B cell tissue, interested especially B cell surface marker is preferentially expressed on the B cell, and can express on precursor B cell and mature B cell.In one embodiment, preferred marker is CD19, and it is based on from the B cell between the differentiation phase of the pedigree in the plasmocyte stage of former/terminal differentiation of pre B cell stage process.

Term used herein " antibody half life " refers to the pharmacokinetic property of antibody, promptly according to the amount of the average lifetime of the antibody molecule of its administration.The antibody half life, can be expressed as the needed time of immunoglobulin (Ig) of removing 50% known quantity from patient's body or its specific part, for example, and when in serum, measuring, that is, and the circulation half life, or in other tissue.Half life, can be different between a kind of immunoglobulin (Ig) or an immunoglobulin like protein and another kind of immunoglobulin (Ig).Usually, the increase of antibody half life causes the increase of mean residence time (MRT) in the antibody administration circulation.

Term " isotype " refers to the classification of antibody.The constant region of antibody does not participate in antigenic combination, but shows various operon functions.According to the aminoacid sequence of CH, given antibody or immunoglobulin (Ig) can be divided into one of five kinds of main immunoglobulin classes: IgA, IgD, IgE, IgG and IgM.The part of these types can be further divided into subclass (isotype), for example: IgG1 (γ 1), IgG2 (γ 2), IgG3 (γ 3) and IgG4 (γ 4), and IgA1 and IgA2.CH corresponding to dissimilar immunoglobulin (Ig)s is known as α, Δ, epsilon, γ and μ respectively.The structure and the three-dimensional configuration of known dissimilar immunoglobulin (Ig).In the type of various human normal immunoglobulins, known only human IgG1, IgG2, IgG3, IgG4 and IgM activating complement.Known person IgG1 and IgG3 mediate ADCC in human body.

As used herein, term " immunogenicity " refers to that mixture can cause immune response (exciting the generation of specific antibody and/or the propagation of specific T-cells).

As used herein, term " antigenicity " refers to that mixture discerned by antibody, maybe can be attached on the antibody, and the induction of immunity reaction.

As used herein, the antigenic overall bonding strength of term " avidity " antibodies (that is: two antibody arms) measures.Antibody affinity can use any known method in this area Ag-Ab connects when being determined at antigen excess dissociates to determine, for example, but be not limited to, use by people such as Gray, J.Virol.Metk, the improving one's methods of 44:11-24 (1993) described indirect fluorescent antibody.

By term " treatment ", " treatment " or " right ... treatment " (or grammatical corresponding term), its severity that refers to the state of an illness of object reduces, or realize improving at least in part or improving and/or at least one clinical symptom, have some to alleviate, alleviate or reduce, and/or in the development of the state of an illness, have inhibition or delay, and/or the morbidity of prevention or delay illness or disease.Therefore, term " treatment ", " treatment " or " right ... " (or grammatical corresponding term) refers to prevention and therapeutic modality in treatment.

As used herein, term " enough amounts " or " capacity " obtain specific effect and refer to an amount of antibody of the present invention or composition, it can effectively produce desired result, described effect be optionally result of treatment (that is: by the treatment significant quantity administration).For example, " enough amounts " or " capacity " can be the amount of effective consumption B cell.

" effective in the treatment " amount used herein is the amount that provides some to alleviate, alleviate and/or reduce at least one clinical symptom.The relevant clinical symptom of illness that the known and available method of the present invention of those skilled in the art is treated.Further, those skilled in the art understand result of treatment and need not to be and maybe can cure the disease completely, as long as can provide some benefits to object.

4. brief description of the drawings

The CD19 of Figure 1A-1E explanation hCD19TG mouse cell lines expresses.Figure 1A shows from hCD19TG (TG-1 ^+/-) people of B cell of mouse and the CD19 of mouse express.Figure 1B shows the CD19 from hCD 19TG mouse ⁺The relative mean density that the people of blood B cell and mouse CD19 express.Fig. 1 C shows from TG-1 ^+/-The CD19 of mouse tissue ⁺The relative density that the hCD19 of B cell and mCD19 express.Fig. 1 D shows that CD19 antibody is from TG-1 ^+/-Mouse blood of mouse and spleen B220 ⁺On the B cell in conjunction with density.Fig. 1 E shows the anti-CD 19 antibodies on 300.19 cells that are attached to hCD19 cDNA transfection.

Fig. 2 A-2D is presented at the consumption of blood in the hCD19TG mouse, spleen and lymphoglandula B cell.Fig. 2 A shows at TG-1 ^+/-Behind contrast (CTL) Antybody therapy of the CD19 of mouse or isotype coupling 7 days, typically come the B cell consumption of autoblood, spleen and lymphoglandula.Fig. 2 C and Fig. 2 D are presented at CD19 (band of filling) that indicates dosage or contrast (empty band) Antybody therapy TG-1 ^+/-Behind the mouse, spleen and lymphoglandula B cell divide other number (± SEM).

Fig. 3 A-3F is described in the anti-CD 19 antibodies treatment consumption of marrow B cell afterwards.Fig. 3 A shows the TG-1 of the four look immunofluorescence dyeings mensuration of analyzing with the stream cell counting ^+/-The typical hCD19 of marrow B cell filial generation and mCD19 express.Fig. 3 B shows that the four look immunofluorescence dyeings analyzed with the stream cell counting are measured, after control antibodies (the 250 μ g) treatment of FMC63 or isotype coupling seven days, and hCD19 in the marrow of hCD19TG mouse ⁺The consumption of cell.Fig. 3 C is presented at control antibodies (250 μ g) the treatment TG-1 of CD19 or isotype coupling ^+/-Mouse seven days, representational B220 in marrow ⁺The B cell consumption.Fig. 3 D is presented at control antibodies (250 μ g) the treatment TG-1 of FMC63 or isotype coupling ^+/-Mouse seven days, the representational B cell subsets of measuring with the trichromatic immunofluorescence dyeing in marrow consumes.According to CD43 express (figure below) further with IgM-B22010 former/the pre B cell segmentation.Fig. 3 E is presented at the control antibodies (250 μ g) of FMC63 or isotype coupling and treated the hCD19TG mouse species seven days, with the representational CD25 of double-colored immunofluorescence dyeing mensuration ⁺The consumption of B22010 pre B cell.Fig. 3 F is presented at FMC63 (band of sealing) or contrast (open band) 〉=3 pairs of littermates was carried out Antybody therapy seven days, is illustrated in number (± SEM) the histogram of pro B lymphocyte (pro-B cell) in the thigh of both sides, pre B cell, jejune and sophisticated B cell.

Fig. 4 A-4C shows peritoneal cavity B cell antagonism CD19 Antybody therapy sensitivity.Fig. 4 A shows by peritoneal cavity CD5 ⁺B220 ⁺B ^IaAnd CD5-B220 ^HiThe people and the mouse CD19 of B2 (routine) B cell express.Fig. 4 B shows use by oneself CD19 (HB12a of 250 μ g, HB12b and FMC63; The B4 of 50 μ g and HD237) TG-1 of antibody or control antibodies (250 μ g) treatment ^+/-The peritoneal cavity B220 of mouse ⁺The consumption of cell.Fig. 4 C is presented at anti-CD19 or control antibodies was treated the hCD19TG mouse seven days, representational CD25 ⁺B220 ⁺B ^IaAnd CD5-B220 ^HiThe consumption of B2 B cell.

The heavy chain VH-D-JH that Fig. 5 A describes the HB12a anti-CD 19 antibodies engages the Nucleotide (SEQ ID NO:1) of sequence and amino acid (the SEQ ID NO:2) sequence of prediction.The heavy chain VH-D-JH that Fig. 5 B describes the HB12b anti-CD 19 antibodies engages the Nucleotide (SEQ ID NO:3) of sequence and amino acid (the SEQ ID NO:4) sequence of prediction.

Fig. 6 A describes the Nucleotide (SEQ ID NO:15) of the sequence of light chain of HB12a anti-CD 19 antibodies and amino acid (the SEQ ID NO:16) sequence of prediction.Fig. 6 B describes the Nucleotide (SEQ ID NO:17) of the sequence of light chain of HB12b anti-CD 19 antibodies and amino acid (SEQ IDNO:18) sequence of prediction.

Fig. 7 A-7B describes the aminoacid sequence of disclosed mouse anti (people) CD19 antibody.Fig. 7 A shows that the heavy chain VH-D-JH that comprises consensus sequence (SEQ ID NO:5), HB 12a (SEQ ID NO:2), 4G7 (SEQ ID NO:6), HB 12b (SEQ ID NO:4), HD37 (SEQ ID NO:7), B43 (SEQ ID NO:8) and FMC63 (SEQ ID NO:9) engages the sequence of sequence.Fig. 7 B shows the light chain VK amino acid sequence analysis of anti-CD 19 antibodies.Consensus sequence (SEQ ID NO:10), HB12a (SEQ ID NO:16), HB12b (SEQID NO:18), HD37 (SEQ ID NO:11), B43 (SEQ ID NO:12), FMC63 (SEQ ID NO:13) and 4G7 (SEQ ID NO:14) have been located.

Fig. 8 A-8C shown the CD19 concentration affects in the body by the efficient of the B cell consumption of anti-CD 19 antibodies.Shown according to behind HB12b (Fig. 8 A) or FMC63 (Fig. 8 B) Antybody therapy (seven days, 250 μ g/ mouse) the B cell consumption of representational blood and spleen in the hCD19TG mouse.Fig. 8 C is presented at from TG-1 ^+/-The blood B220 of mouse ⁺CD19 of antagonism mutually on the B cell and anti-CD20 antibodies are in conjunction with concentration.Fig. 8 D is presented at from TG-1 ^+/-The spleen B220 of mouse ⁺CD19 of antagonism mutually on the B cell and anti-CD20 antibodies are in conjunction with concentration.

Fig. 9 A-9D shows that the consumption by the B cell of anti-CD 19 antibodies treatment is that FcR γ-and monocyte is dependent.Fig. 9 A is at hCD19 TG-1 ^+/-FcR γ ^+/-The B cell consumption of 7 days representational blood and spleen after the treatment of the CD19 of littermate or isotype control antibodies.Fig. 9 B was at the 0th day FcR γ ^-/-The B cell consumption of 7 days blood and tissue after the Antybody therapy of littermate.The hCD19TG-1 that Fig. 9 C consumes at monocyte ^+/-Representational B cell number in the mouse.The B cell consumption of Fig. 9 D seven days blood and tissue after Antybody therapy.

Figure 10 A-10D shows time length and the dose response by the B cell consumption of anti-CD 19 antibodies treatment.Figure 10 A is presented at the 0th day TG-1 by FMC63 or the treatment of isotype control antibodies ^+/-A large amount of blood B220 of mouse ⁺B cell and Thy-1 ⁺The T cell.Figure 10 B-C shows the Antybody therapy representational B cell consumption of organizing in mouse shown in 11,16 and 30 week back Figure 10 A afterwards.Figure 10 D shows the anti-CD 19 antibodies dosage corresponding to the B cell consumption of blood, marrow and spleen.

Figure 11 A-11C shows the CD19 not internalization by antibodies in vivo.Mating control antibodies (250 μ g) interior therapeutic TG-1 with HB12a (Figure 11 A), HB12b (Figure 11 B), FMC63 (Figure 11 C) or isotype ^+/-Cell surface CD19 expresses and the removing of B cell in the mouse.

Figure 12 A-12C shows in the body by anti-CD 19 antibodies bonded CD19 Sa.The TG-1 that treat with FMC63 or isotype coupling control antibodies (250 μ g) Figure 12 A display body inherence ^+/-The removing of B cell in the mouse.Figure 12 B shows that FMC63 Antybody therapy (250 μ g) makes the antibody combining site on the hCD19 saturated in administration within an hour.Figure 12 C shows that HB12b anti-CD 19 antibodies treatment (250 μ g) is as within an hour making antibody combining site hCD19 on saturated in administration at Figure 12 B determined.

Figure 13 A-13B shows that the anti-CD 19 antibodies treatment is at TG-1 ^+/-Reduced the level of serum immune globulin and autoantibody in the mouse.Figure 13 A describes the level of serum immune globulin, and Figure 13 B is described in the level of anti-CD 19 antibodies treatment anti-dsDNA, anti-ssDNA and anti-histone autoantibody afterwards.

Figure 14 A-14B shows that anti-CD 19 antibodies treatment blocked TG-1 ^+/-The immunne response that causes by body fluid in the mouse.Mouse with the DNP-KLH immune antibody treatment of the DNP-Ficoll of TNP-LPS, Figure 14 B of Figure 14 A and Figure 14 C-14D.Before first immunisation the 0th day 7 days (A-C) or 14 days afterwards (D), with FMC63 (airtight circle) or contrast (open circle) antibody (250 μ g) treatment littermate.

Figure 15 shows and adds the treatment of anti-CD19 and anti-CD20 antibodies simultaneously.

Figure 16 show (i.p.) and vein (i.v.) administration in subcutaneous (s.c.), the abdomen of anti-CD 19 antibodies effectively the consumer internal recycle with organize in the B cell.

The treatment of Figure 17 A-17B anti-CD 19 antibodies has stoped hCD19 in the body ⁺Lymphadenomatous growth (Figure 17 A), and increased survival rate (Figure 17 B).

5. detailed description of the invention

The present invention relates to be used for the treatment of human body B cell disease and disorderly immunotherapeutic composition and method, for example, but be not limited only to, use the therapeutic antibodies in conjunction with CD19 antigen, the CDCC (ADCC) of preferred people's antibody dependent cellular mediation is treated the B cell malignancies. The present invention relates to comprise the pharmaceutical composition of the IgG1 people, humanized or chimeric or IgG3 people's isotype anti-CD 19 antibodies. The invention still further relates to comprise IgG2 or IgG4 people's isotype the people's or humanized anti-CD 19 antibodies, preferably mediate the pharmaceutical composition of human ADCC. In certain embodiments, the invention still further relates to the pharmaceutical composition that comprises anti-CD 19 antibodies monoclonal human, humanized or chimeric that available methods known in the art are produced.

Describe treatment be diagnosed as by the B cell and and prescription and the dosage regimen of the treatment of the human patients of precursor-derived B cell malignancies, described B cell and and precursor-derived B cell malignancies, include but are not limited to acute lymphoblastic leukemia (ALL), hodgkin's lymphomas, non-Hodgkin′s lymphomas, B cell chronic lymphocytic leukemia (CLL), Huppert's disease, follicular lymphoma, lymphoma mantle cell, front-lymphocytic leukemia, hairy cell leukemia, common acute lymphocytic leukemia and some non-acute lymphoblastic leukemia.

5.1 the generation of anti-CD 19 antibodies

5.1.1 polyclone anti-CD 19 antibodies

Polyclonal antibody preferably by with related antigen and repeatedly subcutaneous (s.c.) or abdomen interior (i.p.) injection of adjuvant, produces in animal body. With bifunctional reagent or derivative reagent, for example dimaleoyl imino benzoyl (maleimidobertzoyl) sulfenyl succinimide ester (by cysteine residues in conjunction with), N-hydroxy-succinamide (passing through lysine residue), glutaraldehyde, succinyl oxide, SOCl₂, with related antigen and immune species immunogen protein, for example key hole keyhole limpet hemocyanin, seralbumin, bovine thyroglobulin or soybean trypsin inhibitor combination may be useful.

Come immune animal with antigen, immune conjugate or derivative, for example 100 μ g or 5 μ g protein or conjugate (respectively for rabbit or mouse) are mixed with the complete Freund's adjuvant of 3 times of amounts, solution is injected in the corium on a plurality of positions. After one month, contain the peptide of 1/5 to 1/10 commercial weight or the incomplete Freund's adjuvant of conjugate for animal hypodermic injection on a plurality of positions. After 7 to 14 days, to the animal blood drawing, measure the antibody titer of its serum. To the animal booster immunization until titre is constant. Preferably, with identical but by different protein bound and/or come the antigen conjugate of combination to the animal booster immunization by different cross-linking reagents. Also can in the recombinant cell culture thing, the form with protein fusions prepare conjugate. Condensate for example alum also is fit to strengthen immune response.

5.1.2 monoclonal anti-CD 19 antibodies

Monoclonal anti-CD 19 antibodies of the present invention shows the binding specificity with people CD19 antigen, and the preferred ADCC of Mediated Human. These antibody can be used multiple technologies known in the art, comprise utilizing hybridoma, restructuring and display technique of bacteriophage or its to make up to obtain. Antibody is high degree of specificity, and it is for single antigen site. In addition, compare from traditional (polyclone) antibody preparation that generally comprises for different determinants (epitope), each monoclonal antibody is for the single determinant on the people CD19 antibody. For example, can be with people such as Kohler according to monoclonal antibody used in the present invention, Nature, the hybridoma method that 256:495 (1975) describes first prepares it and can be used for producing the mouse-anti body and (or derive from the antibody of other non-human mammal, such as mouse, goat, sheep, ox, camel etc. ) or derive from transgenic animals people's antibody (referring to, U.S. Patent number 6,075,181,6,114,598,6,150,584 and 6,657,103). Optionally, can with recombinant DNA technology prepare monoclonal antibody (referring to, for example: U.S. Patent number 4,816,567), and described monoclonal antibody comprises chimeric and humanized antibody. " monoclonal antibody " is also available such as people such as Clackson, Nature, and the people such as 352:624-628 (1991) and Marks, J.Mol Biol, the described method of 222:581-597 (1991) is separated from phage antibody library.

Can come the preparation engineering anti-CD 19 antibodies with any method known in the art, include but are not limited to the improvement of following method and these methods. Large-scale high yield production relates generally to cultivate the host cell that produces the through engineering approaches anti-CD 19 antibodies, and reclaims anti-CD 19 antibodies from the host cell culture.

5.1.3 hybridoma technology

Useful hybridoma technology prepares monoclonal antibody, comprising known technology in this area and the instruction, such as people such as Harlow, Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed, 1988); The people such as Hammerling are at Monoclonal Antibodies and T Cell Hybridomas, among the 563-681 (Elsevier, N.Y. 1981) (described list of references is incorporated into herein as a reference with its integral body). For example, in hybridoma method, with mouse or other suitable host animal, for example hamster or macaque immunity maybe can produce the lymphocyte that carries out the antibody that specificity is combined with the albumen that is used for immunity to obtain producing. Alternatively, can be at external immunological lymphocyte. Then utilize suitable flux to mix with the myeloma cell in lymphocyte, for example polyethylene glycol produces hybridoma (Goding, Monoclonal Antibodies:Principles and Practice, 59-103 (Academic Press, 1986).

Also cultivate so hybridoma of preparation at suitable inoculation of medium, described medium optimization comprises one or more and suppresses the not parental generation myeloma cell's of fusion growth or the material of survival rate. For example, if the myeloma cell of parental generation lacks hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium of hybridoma generally will comprise hypoxanthine, aminopterin and thymidine (HAT medium) so, and this material hinders the growth of HGPRT-deficient cell.

Preferred efficient myeloma cell of merging uses the antibody produced cell of selecting to keep and stablize high-caliber antibody generation, and described cell is responsive to medium, for example the HAT medium. Among these cells, preferred myeloma cell line is rat bone marrow tumour cell system, for example derive from Salk institute Cell Distribution Center, San diego, CA is among the MOPC-21 and MPC-11 mouse tumors that USA obtains, and from American Type Culture Collection, Rockville, the clone of the SP-2 that MD, USA obtain or X63-Ag8.653 cell. Human myeloma and mouse people heteromyeloma clone also are described to produce people's monoclonal antibody (Kozbor, J. Immunol, 133:3001 (1984); The people such as Brodeur, Monoclonal Antibody Production Techniques and Application, 51-63 (Marcel Dekker, Inc, New York, 1987).

For producing the monoclonal antibody for people CD19 antigen, measure the culture medium of Growth of Hybridoma Cell. Preferably, with immune precipitation or by external combination test, for example the binding specificity with the monoclonal antibody of hybridoma production is checked in radioimmunoassay (RIA) or EUSA (ELISA).

After having identified that antibody that hybridoma produces has specificity, affinity and/or the activity of requirement, can be by this cell of method subclone of limiting dilution, and according to standard method cultivation (Goding, Monoclonal Antibodies:Principles and Practice, 59-103 (Academic Press, 1986)). The appropriate culture medium that can reach this purpose comprises, for example: D-MEM or RPMI1640 culture medium. In addition, hybridoma may grow into belly cavity tumor in animal body.

To suitably be separated from the culture medium, seroperitoneum or the serum that obtain by traditional immunoglobulin purification method by the oozy monoclonal antibody of subclone, described purification process is a-protein-Ago-Gel, hydroxyapatite chromatography method, gel electrophoresis, dialysis or affinity chromatography for example.

5.1.4 recombinant DNA technology

Utilize conventional program (for example: utilize can specific binding coding anti-CD 19 antibodies heavy chain and the oligonucleotide probe of the gene of light chain), separate and the invention of order-checking code book in the DNA of anti-CD19 antibody. With the source of hybridoma as preferred this DNA. In case separate, DNA can be put into expression vector, then it is transfected into host cell, for example Bacillus coli cells, monkey COS cell, Chinese hamster ovary (CHO) cell or no longer produce the myeloma cell of immunoglobulin (Ig) are to realize anti-CD 19 antibodies synthetic in the restructuring host cell.

In display technique of bacteriophage, showed the functional antibodies domain on the surface of bacteriophage particles, it is with their polynucleotide sequence of coding. Especially, the DNA sequence of amplification coding VH and VL domain from animal cDNA library (for example: the people of infected tissue or mouse cDNA library). Utilize will the encode dna sequence dna of VH and VL domain of PCR to be binned in the scFv connexon, and be cloned into phagemid vector. The carrier electricity is transformed among the E.coli, and infects E.coli with helper phage. Bacteriophage used in the method is generally filobactivirus, and it comprises fd and M13, and VH and common reorganized being fused among phage gene III or the gene VIII of VL domain. The bacteriophage of expressing the antibody binding structural domain that is attached to specific antigen is selected or identified to available antigen, for example: with antigen or the combination of mark or capture antigen on the surface of solids or the pearl. The example that can be used to make the display technique of bacteriophage of antibody of the present invention comprises the people such as Brinkman, 1995, J.Immunol.Methods, 182:41-50; The people such as Ames, 1995, J.Immunol.Methods, 182:41-50; The people such as Ames, 1995, J.Immunol.Methods, 184:177-186; The people such as Kettleborough, 1994, Eur.J.Immunol, 24:952-958; The people such as Persic, 1997, Gene, 187:9-18; The people such as Burton, 1994, Advances in Immunology, 57:191-280; International application no PCT/GB91/O1134; International publication number WO 90/02809, WO 91/10737, and WO 92/01047, and WO 92/18619, and WO 93/11236, and WO 95/15982, WO 95/20401 and WO 97/13844; 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; Each document is wherein incorporated into herein as a reference with its integral body.

As described in above-mentioned list of references, after bacteriophage is selected, separable antibody coding region from bacteriophage, and be used for producing complete antibody, the Fab that comprises people's antibody or any other needs, and express among the host who wants that what is the need in office, comprise mammalian cell, insect cell, plant cell, yeast and bacterium, as described below. Also can use recombinant production Fab, Fab ' and F (ab ') with methods known in the art₂The technology of fragment, for example PCT publication number WO 92/22324; The people such as Mullinax, 1992, BioTechniques, 12 (6): 864-869; The people such as Sawai, 1995, AJRI, the people such as 34:26-34 and Better, 1988, Science, 240:1041-1043 (described list of references is incorporated herein by reference with its integral body).

In further embodiment, can be from the antibody phage library separation antibody, described antibody phage library is to use the people such as McCafferty, Nature, the described technology of 348:552-554 (1990) produces. The people such as Clackson, Nature, 352:624-628 (1991). The people such as Marks, J.Mol.Biol, 222:581-597 (1991) have described respectively with phage library and have separated mouse and people's antibody. Chain is replaced the preparation can be used for high affinity (nm scope) people's antibody (people such as Marks, Bio/Technology, 10:779-783 (1992)), also have combination to infect and the interior restructuring of body, as the strategy that makes up extensive phage library people such as (, Nuc.Acids.Res.21:2265-2266 (1993)) Waterhouse. Like this, the alternative traditional monoclonal antibody hybridoma technology of the method is used for the separation of anti-CD 19 antibodies.

For producing complete antibody, the flanking sequence that comprises VH or VL nucleotide sequence, restriction site and protection restriction site can be used for increasing VH or VL sequence in the scFv clone. Utilize known to those skilled in the art cloning process, the VH domain of pcr amplification can be cloned in the carrier of expressing the VH constant region, for example: people γ 4 constant regions, the VL domain of PCR amplification can be cloned in the carrier of expressing the VL constant region, for example: human kappa or lamba constant region. The carrier of preferably, expressing VH or VL domain comprises cloning site, constant region and the selected marker of EF-1 α promoter, secretion signal, variable region, for example selected marker of neomycin. Also VH and VL domain can be cloned in the carrier of the essential constant region of expression. Then utilize technology well known by persons skilled in the art, heavy chain conversion carrier and light chain conversion carrier cotransfection are entered in the clone, produce and (for example: stable or unsettled clone IgG) express full length antibody.

But also modifying DNA, for example the coded sequence of employment heavy chain and light chain constant domain replaces mouse sequence (U.S. Patent number 4,816,567 of homology; The people such as Morrison, Proc.Natl.Acad.Sci.USA, 81:6851 (1984)), or be connected on the immunoglobulin coding sequence by all or part of coded sequence of covalency with the NIg polypeptide.

5.1.5 chimeric antibody

Anti-CD 19 antibodies herein comprises chimeric antibody (immunoglobulin (Ig)) especially, wherein the part of heavy chain and/or light chain is equal to or comes from from the corresponding sequence of individually defined thing species or genus in the antibody of specific antibodies class or subclass together, and another part of chain is equal to or with coming from from another species or belonging in the antibody of another antibody class or subclass, and the corresponding sequence in this antibody-like fragment, as long as this antibody or antibody fragment have shown the biologically active (U.S. Patent number 4 of needs, 816,567; The people such as Morrison, Proc.Natl.Acad.Sci.USA, 81:6851-6855 (1984)). The chimeric antibody of research comprises " Primates " antibody herein, its comprise from non-human primates (for example: gerontogeous monkey, such as baboon, rhesus macaque or machin) variable domains antigen binding sequence and the sequence (U.S. Patent number 5,693,780) of human constant region.

5.1.6 humanized antibody

Can prepare humanized antibody with various technology known in the art, include but are not limited to CDR-replace (referring to, for example: european patent number EP 239,400; International publication number WO 91/09967; And U.S. Patent number 5,225,539,5,530,101 and 5,585,089, each document is wherein incorporated into herein as a reference with its integral body), veneer or surface replacement (referring to, for example: european patent number EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology28 (4/5): 489-498; The people such as Studnicka, 1994, Protein Engineering Protein Engineering, 7 (6): the people such as 805-814 and Roguska, 1994, PNAS, 91:969-973, each document is wherein incorporated into herein as a reference with its integral body), the chain replacement (referring to, for example: U.S. Patent number 5,565,332, it is incorporated into herein as a reference with integral body), and disclosed technology, for example: U.S. Patent number 6,407,213, United States Patent (USP) number 5,766,886, international publication number WO 9317105, the people such as Tan, J.Immunol, 169:1119-25 (2002), the people such as Caldas, Protein Eng.13 (5): 353-60 (2000), the people such as Morea, Methods, 20 (3): 267-79 (2000), the people such as Baca, J.Biol.Chem.272 (16): 10678-84 (1997), the people such as Roguska, Protein Eng, 9 (10): 895-904 (1996), the people such as Couto, Cancer Res.55 (23 Supp): 5973s-5977s (1995), the people such as Couto, Cancer Res.55 (8): 1717-22 (1995), Sandhu JS, Gene, 150 (2): the people such as 409-10 (1994) and Pedersen, J.Mol.Biol, 235 (3): 959-73 (1994), and each document is wherein incorporated into herein as a reference with its integral body. Usually, use the corresponding residue from the CDR donor antibody to replace the framework residue of framework region, preferably promote the combination of antigen. Identify these framework substituents with technology known in the art, for example: identify for antigen in conjunction with important framework residue by the interaction mode of CDRs and framework residue, and sequence alignment is to identify at the unusual framework residue of ad-hoc location. (referring to, such as: the people such as Queen, U.S. Patent number 5,585,089; With the people such as Riechmann, 1988, Nature, 332:323, it is incorporated into herein as a reference with integral body).

Humanized anti-CD 19 antibodies has one or more amino acid residues of introducing from inhuman source. These inhuman amino acid residues are commonly called " input " residue, its general acquisition from " input " variable region. Therefore, humanized antibody comprises from one or more CDR of non-human immunoglobulin molecule with from people's framework region. Be known in the art the humanization of antibody, and can operate according to Winter and its colleagues' method that (321:522-525 (1986) for the people such as Jones, Nature; The people such as Riechmann, Nature, 332:323-327 (1988); The people such as Verhoeyen, Science, 239:1534-1536 (1988)), being the corresponding sequence of people's antibody by CDRs or the replacement of CDR sequence with rodent, i.e. (EP 239,400 in CDR-replacement; PCT publication number WO 91/09967; And U.S. Patent number 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, its content is incorporated into herein as a reference with its integral body). In this humanization chimeric antibody, be less than substantially complete people's variable domains and replaced by non-human species's corresponding sequence. In fact, humanized antibody is typical people's antibody, and some of them CDR residue is replaced in similar site by the antibody of rodent with some possible FR residues. The humanization of anti-CD 19 antibodies also can realize that (EP 592,106 by veneer or surface replacement; EP 519,596; Padlan, 1991, Molecular Immunology 28 (4/5): 489-498; The people such as Studnicka, Protein Engineering, 7 (6): 805-814 (1994); And the people such as Roguska, PNAS, 91:969-973 (1994)) or chain replace (U.S. Patent number 5,565,332), he its content is incorporated into herein as a reference with its integral body.

For the preparation of the selection of the human variable region (comprising light chain and heavy chain) of humanized antibody, be in order to reduce antigenicity. According to so-called " best fit " method, the variable region sequences of rodent antibody is screened from the Full-text Database of known people's variable region sequences. Then, will be close to the human sequence of rodent sequence as the human framework that is used for humanized antibody (people such as Sims, J.Immunol, 151:2296 (1993); The people such as Chothia, J.Mol.Biol, 196:901 (1987), its content is incorporated into herein as a reference with its integral body). Another method is used the specific framework from the consensus sequence of everyone antibody-like of the light chain of specific subclass or heavy chain. Same framework can be used for several different Humanized anti-cd 19 antibodies (people such as Carter, Proc.Natl.Acad.Sci.USA, 89:4285 (1992); The people such as Vresta, J.Immunol, 151:2623 (1993), its content is incorporated into herein as a reference with its integral body).

Anti-CD 19 antibodies can by humanization, make its maintenance to high affinity and other the good biological characteristics of CD19. According to an aspect of the present invention, by analyze the process of parental generation sequence and various notional humanization products with the threedimensional model of parental generation and humanized sequence, prepare humanized antibody. Three-dimensional immunoglobulin (Ig) model is usually obtainable, and is well-known to those skilled in the art. The possible three-dimensional conformation structure of candidate's immunoglobulin sequences that the diagram of available computers program and demonstration are chosen. Check that these demonstrations can analyze may acting on that residue plays in the function of candidate's immunoglobulin sequences, that is: analyzing influence candidate immunoglobulin (Ig) is in conjunction with the residue of the ability of CD19. Like this, can from acceptor and list entries, select and in conjunction with the FR residue, in order to obtain the antibody feature that needs, for example to the affinity of the increase of CD19. Usually, the CDR residue directly and to a great extent participates in impact to the combination of antigen.

The antibody of " humanization " keeps the antigentic specificity similar to original antibody, that is: in the present invention, and in conjunction with the ability of people CD19 antigen. Yet, utilize certain humanized method, " orthogenesis " method of use, can increase antibody in conjunction with affinity and/or the specificity of people CD19 antigen, the people such as described " orthogenesis " method such as Wu, J.Mol.Biol, 294:151 (1999), each document is wherein incorporated into herein as a reference with its integral body.

5.1.7 people's antibody

In order in people's body, to use antibody, preferred end user's antibody. In order to treat human subjects, need especially complete people's antibody. Can prepare with the whole bag of tricks known in the art people's antibody, these methods comprise that utilization mentioned above obtains the display technique of bacteriophage from the antibody library of human immunoglobulin(HIg) sequence, comprises the improvement to these technology. Simultaneously referring to, U.S. Patent number 4,444,887 and 4,716,111; With the open text WO 98/46645 of PCT, WO 98/50433, and WO 98/24893, and WO 98/16654, and WO 96/34096, WO 96/33735 and WO 91/10741, and each document is wherein incorporated into herein as a reference with its integral body. People's antibody also can be such antibody, coded heavy chain and the light chain of nucleotide sequence that it is originated from one or more human DNAs with acquisition.

Also available can not the endogenous immunoglobulin (Ig) of expressive function, but the transgenic mice that can express the human immunoglobulin gene prepares people's anti-CD 19 antibodies. For example: can with people's heavy chain and light chain immune protein gene complex at random or by homologous recombination in the embryonic stem cell of mouse. Optionally, except people's heavy chain and light chain gene, people variable region, constant region and difference section can be imported in the embryonic stem cell of mouse. After the site imported human immunoglobulin(HIg), the heavy chain of mouse and light chain immunoglobulin gene may be respectively or are shown as simultaneously nonfunctional by homologous recombination. For example: described, heavy chain bonding pad (JH) gene of the antibody of chimeric and mouse germ line mutation is carried out RT-PCR, caused the fully inhibition that endogenous antibody is produced. The embryonic stem cell amplification that changes and microinjection are entered blastocyst prepare gomphosis mouse. Then make the gomphosis mouse breeding, produce the offspring of isozygotying who expresses people's antibody. With the antigen of selecting transgenic mice is carried out immunity with usual way, described antigen for example: polypeptide of the present invention whole or a part of. Available traditional hybridoma technology obtains the anti-CD 19 antibodies for people CD19 antigen from the transgenic mice of immunity. The all human immunoglobulin(HIg) transgenosis of transgenic mice were reset in the B Cell Differentiation phase, had experienced again afterwards classification conversion and somatic mutation. Therefore, use this technology, may prepare upper useful IgG, IgA, IgM and the IgE antibody for the treatment of, include but are not limited to IgG1 (γ 1) and IgG3. To the general introduction for preparing people's antibody with this technology, referring to Lonberg and Huszar (Int.Rev. Immunol, 13:65-93 (1995)). To this technology of preparation people's antibody and human monoclonal antibodies and discussing in detail of the technical scheme for preparing such antibody, referring to, for example: PCT publication number WO 98/24893, WO 96/34096 and WO 96/33735; And 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, each document is wherein incorporated into herein as a reference with its integral body. In addition, Abgenix for example, the companies such as Inc. (Freemont, CA) and Genpharm (San Jose, CA) can provide with similar techniques mentioned above people's antibody for selected antigen. Concrete discussion for the transfer of the people's fetal immune globulin gene in the mouse of germ line mutation, to cause the generation of people's antibody of antigen stimulation, referring to, such as: the people such as Jakobovits, Proc.Natl.Acad.Sci.USA, 90:2551 (1993); The people such as Jakobovits, Nature, 362:255-258 (1993); The people such as Bruggermann, Year in Immunol, the people such as 7:33 (1993) and Duchosal, Nature, 355:258 (1992).

Also can from phage display library, obtain people's antibody (people such as Hoogenboom, J.Mol.Biol.227:381 (1991); People such as Marks, J.Mol.Biol.222:581-597 (1991); People such as Vaughan, Nature Biotech.14:309 (1996)).Display technique of bacteriophage people such as (, Nature, 348:552-553 (1990)) McCafferty can be used for by all from the gene in the immunoglobulin variable of immune donor (V) district not at produced in vitro human antibodies and antibody fragment.According to this method, antibody V district's gene clone that will be in framework is to the main or less important coat protein gene of filobactivirus, for example: M13 or fd, and on the bacteriophage particles surface, show as the functional antibodies fragment.Because filamentous particle comprises single stranded DNA copy of phage genome, the selection of making according to the antibody function characteristic also can cause the encoding selection of gene of the antibody of showing these characteristics.Therefore, phage has been simulated some characteristics of B cell.Available various forms carries out phage display; For its general introduction, referring to, for example: Johnson, Kevin S. and Chiswell, David J.Current Opinion in StructuralBiology 3:564-571 (1993).Some sources of V-gene fragment can be used for phage display.People such as Clackson, Nature, 352:624-628 (1991) has not isolated various anti-oxazolone antibody the small-sized combinatorial library at random of the V gene of immune mice spleen certainly from acquisition.Can make up from whole integral parts without the V gene of people's donor of immunity, and can be according to people such as Marks, people such as J.Mol.Biol.222:581-597 (1991) or Griffith, the method that EMBOJ.12:725-734 (1993) describes is thoroughly isolated at the various antigens antibody of (comprising autoantigen).Simultaneously referring to, U.S. Patent number 5,565,332 and 5,573,905, each document is wherein incorporated into herein as a reference with its integral body.

Also available external activatory B cells produce people antibody (referring to, United States Patent (USP) 5,567,610 and 5,229,275, each document is wherein incorporated into herein as a reference with its integral body).Also available hybridoma technology for example, but is not limited only at produced in vitro people antibody, the method (Methods Enzymol, 121:140-167 (1986)) that people such as Roder describe.

5.1.8 change/antibody of sudden change

Anti-CD 19 antibodies in moiety of the present invention and the method can be the antibody of sudden change.As used herein, " antibody mutation " or " antibody of change " refers to the aminoacid sequence variant of anti-CD 19 antibodies, and wherein one or more amino-acid residues of anti-CD 19 antibodies are modified.Change to the anti-CD 19 antibodies aminoacid sequence comprises: for improving the modification that antibody is done sequence its antigenic avidity or close antigenicity, and/or for improving the modification that effector function antagonist Fc partly does.Can modify any known anti-CD 19 antibodies or the determined anti-CD 19 antibodies of description that Click here.Antibody certainty and the known anti-CD 19 antibodies that changes like this has sequence identity or the similarity less than 100%.In preferred embodiments, the antibody that changes has amino acid sequences with anti-CD 19 antibodies heavy chain or light chain at least 25%, 35%, 45%, 55%, 65% or 75% the identity or the aminoacid sequence of similarity is arranged, more preferably at least 80%, more preferably at least 85%, more preferably 90%, most preferably at least 95%.In preferred embodiments, the antibody that changes has aminoacid sequence with heavy chain CDR1, the CDR2 of anti-CD 19 antibodies or CDR3 at least 25%, 35%, 45%, 55%, 65% or 75% the identity or the aminoacid sequence of similarity is arranged, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95%.In preferred embodiments, the antibody of change has kept the binding ability of people CD19.In certain embodiments, anti-CD antibody of the present invention comprises aminoacid sequence with SEQ IDNO:2 (Fig. 5 A) about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or the heavy chain of higher identity, and the aminoacid sequence of described SEQ ID NO:2 (Fig. 5 A) is corresponding with HB12a heavy chain institute.In certain embodiments, anti-CD 19 antibodies of the present invention comprises aminoacid sequence with SEQ ID NO:4 (Fig. 5 B) about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or the heavy chain of higher identity, and the aminoacid sequence of described SEQ ID NO:4 (Fig. 5 B) is corresponding with HB12b heavy chain institute.In certain embodiments, anti-CD 19 antibodies of the present invention comprises aminoacid sequence with SEQ ID NO:16 (Fig. 6 A) about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or the light chain of higher identity, and the aminoacid sequence of described SEQ ID NO:16 (Fig. 6 A) is corresponding with HB12a heavy chain institute.In certain embodiments, anti-CD 19 antibodies of the present invention comprises aminoacid sequence with SEQ ID NO:18 (Fig. 6 B) about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or the light chain of higher identity, and the aminoacid sequence of described SEQ ID NO:16 (Fig. 6 A) is corresponding with HB12b heavy chain institute.The hybridoma that produces HB12a and HB12b anti-CD 19 antibodies carries out preservation with ATCC preserving number PTA-6580 and PTA-6581.

Identity or similarity for this sequence are defined as herein: after carrying out sequence alignment and introducing breach, in the candidate sequence with identical to the anti-CD 19 antibodies residue (being identical residue) or similar (promptly based on common side chain characteristic, from amino-acid residue on the same group, see below) the shared per-cent of amino-acid residue, if desired, obtain the sequence identity of largest percentage.Do not think the N-end, the plug-in effect in terminal or inner expansion, deletion or the antibody sequence outside the variable region of C-the identity or the similarity of sequence.

" % identity " as known in the art is measuring of concerning between two polynucleotide or two polypeptide, determines by their sequence relatively.Two sequences that usually, compare should be arranged as and obtain most relevance between the sequence.Measure the arrangement of two sequences, determine the accurate number of corresponding amino acid between two sequences or Nucleotide, divided by this number, and multiply by 100, just obtained the % value of identity with the total length of arranging.The % value of this identity can be determined by the total length of the sequence of comparing, it is particularly suitable for the sequence of identical or closely similar length, and height homologous sequence, or can determine that it is more suitable in the sequence of unequal length or has sequence than low homology by the length of shorter definition.

For example: the clustalw software under the available Unix operating system comes collating sequence, generation is the file of extension name with " .aln ", this file is imported Bioedit program (Hall, T.A.1999, BioEdit:a user-friendly biological sequence alignment editor and analysisprogram for Windows 95/98/NT.Nucl.Acids.Symp.Ser.41:95-98), open this .aln file with it.In the Bioedit form, can select independent sequence (each two), and arrange them.Compare whole sequence with this method.

Be known in the art the method for the identity of more two or more sequences.For example: Wisconsin sequential analysis routine package 9.1 editions (people such as Devereux J., Nucleic Acids Res.12:387-395,1984, obtain from Genetics Computer Group, Madison, WI USA) provides program.Can use mathematical algorithm to finish the mensuration of identity per-cent between two sequences.For example: can use BESTFIT and GAP program measure two between the polynucleotide % identity and the % identity between two peptide sequences.BESTFIT uses " local homology " algorithm of Smith and Waterman (Advances in Applied Mathematics, 2:482-489,1981), and finds the best single district of similarity between two sequences.BESTFIT is more suitable for comparison length different two polynucleotide or two peptide sequences, and the shorter sequence of this program supposition has been represented the part of longer sequence.Compare, GAP arranges two sequences finding " maximum comparability " according to the algorithm of Neddleman and Wunsch (J.Mol.Biol.48:443-354,1970).GAP is more suitable in the sequence of the length that relatively is roughly the same, and wants arrangement that total length is carried out.The parameter of using in each program " Gap weight " and " length weight " are preferably respectively: for polynucleotide is 50 and 3, is 12 and 4 for polypeptide.Preferably when two sequences that are compared are optimal arrangement, measure % identity and similarity.

Other programs of identity and/or similarity, for example program (Karlin﹠amp of BLAST class between the also known in the art mensuration sequence; Altschul, 1990, Proc.Natl.Acad.Sci.USA, 87:2264-2268 is as Karlin﹠amp; Altschul, 1993, Proc.Natl.Acad.Sci.USA, 90:5873-5877 improves, acquisition from national bioinformation center (National Center forBiotechnology Information, NCB), Bethesda, MD, USA, and the homepage by NCBI Www.ncbi.nlm.nih.govEnter).The demonstrated non-limitative example of the preferred mathematical algorithm that is used for two sequences of comparison of these programs.With people such as this algorithm substitution Altschul, 1990, the NBLAST of J.Mol.Biol.215:403-410 and XBLAST program.Available NBLAST program is carried out the BLAST nucleotide search, score=100, and speech is long by=12, to obtain all or part of the nucleic acid molecule homologous nucleotide sequence with code book invention anti-CD 19 antibodies.Available XBLAST program is carried out the BLAST protein search, score=50, and speech is long by=3, to obtain and protein molecule homologous aminoacid sequence of the present invention.For the arrangement that obtains breach is used for the purpose of comparison, can use people such as Altschul, 1997, the described Gapped BLAST of Nucleic Acids Res.25:3389-3402.Optionally, can use PSI-Blast to carry out repeat search, measure the distance relation between the molecule (Id.).When using BLAST, GappedBLAST, PSI-Blast program, can use separately program () default parameter for example: XBLAST and NBLAST.Referring to, http://www.ncbi.nlm.nih.gov.Another preferred non-limitative example that is used for the mathematical algorithm of comparative sequences is Myers and Miller, 1988, and the algorithm of CABIOS 4:11-17.This algorithm is imported ALIGN program (2.0 editions), and described program is the part of GCG sequence alignment software package.When the ALIGN program is used for the comparing amino acid sequence, use PAM120 weight residue table, notch length deduction of points 12, breach deduction of points 4.

Another non-limitative example of the program of identity and/or similarity is FASTA (Pearson W.R. and Lipman DJ.Proc.Nat.Acad.Sci.USA between the mensuration sequence as known in the art, 85:2444-2448,1988, as the part acquisition of Wisconsin sequence analysis software bag).Preferably with BLOSUM62 aminoacid replacement matrix (Henikoff S. and Henikoff J.G.Proc.Nat.Acad.Sci.USA, 89:10915-10919,1992) be used for peptide sequence relatively, described peptide sequence at first is converted to aminoacid sequence with nucleotide sequence before relatively being included in comparison.

Another non-limitative example of the identity between the mensuration aminoacid sequence as known in the art and/or the program of similarity is SeqWeb software (GCGWisconsin software package based on web interface: the Gap program), the default algorithm of its service routine and parameter setting: blosum62, breach weight 8, length weight 2.

The available method that is similar to aforesaid method is measured two identity per-cents between the sequence, is with or without the breach of allowing.When calculating identity per-cent, the general accurately coupling of calculating.

Measure the polynucleotide of inquiry or the % identity of peptide sequence according to polynucleotide of the present invention or the preferred service routine BESTFIT of peptide sequence, the sequence of inquiry and contrast is in optimal arrangement, and program parameter is located at default value.

Be mutagenic antibody, one or more amino acid changes (for example: replace) imported the hypervariable region of one or more species dependency antibody.Optionally, or in addition, one or more changes (for example: replace) of framework region residue are imported anti-CD 19 antibodies, it causes the antibody mutation body to improve for the antigenic binding affinity from second mammalian species.The example of the framework region residue that changes comprises those direct non-covalent bonded antigens (people such as Amit, Science, 233:747-753 (1986)); In conjunction with/influence the structure (people such as Chothia, J.Mol.Biol.196:901-917 (1987)) of CDR; And/or the boundary (EP239400B1) of participation VL-VH.In certain embodiments, the change of one or more this framework region residues causes antibody for the enhancing from the antigenic binding affinity of second mammalian species.For example: have about one in embodiment of the present invention and arrive about five framework residues change.Sometimes, this may enough produce and be fit to the antibody mutation thing that latency test is used, even it does not have the hypervariable region residue to be changed.Yet in general the antibody of Xiu Gaiing comprises other hypervariable region change.

The hypervariable region residue that changes can be a random variation, and especially it is such at anti-CD 19 antibodies for the antigenic initial binding affinity from second mammalian species, so that the antibody of the change that produces at random can be screened easily.

Effective ways that produce the antibody of this change are known as " alanine scanning mutagenesis " (" alanine scanning mutagenesis ") (Cunningham and Wells, Science, 244:1081-1085 (1989))., replace one or more hypervariable regions residue herein with L-Ala or poly-alanine residue, with influence amino acid with from the antigenic interaction of second mammalian species.Then, by replacing the site or importing additional or other sudden change improves the hypervariable region residue that substituent is shown the function susceptibility at replacing the site.Therefore, when having pre-determined the site that imports the aminoacid sequence change, the character of mutant just needn't pre-determine.According to described herein, screen the Ala-mutant of producing in this way according to its biological activity.

The another kind of method that produces this change antibody relates to avidity slaking (people such as Hawkins, people such as J.Mol.Biol.254:889-896 (1992) and Lowman, Biochemistry, 30 (45): 10832-10837 (1991)) of using phage display.Briefly, the site, several hypervariable region that suddenlys change (for example: 6-7 site), produce all possible aminoacid replacement thing on each site.With the antibody mutation body that produces like this with the unit price form from the filobactivirus particle, display as fusions in that each particle is inner with the gene III product of M13 packing.According to disclosed herein, again (for example: binding affinity) screen the mutant of phage display according to its biological activity.

The sudden change of antibody sequence can comprise replacement, disappearance, comprise inner disappearance, add, comprise the interpolation that produces fusion rotein, or aminoacid sequence is inner and/or guard replacement near the amino-acid residue of aminoacid sequence, but it causes " silence " to change, and wherein changes the anti-CD 19 antibodies that produces equivalence on the function.Can prepare conservative aminoacid replacement thing based on polarity, electric charge, solubility, hydrophobicity, wetting ability and/or the amphiphilic character of the residue that relates to.For example: nonpolar (hydrophobic) amino acid comprises L-Ala, leucine, Isoleucine, Xie Ansuan, proline(Pro), phenylalanine, tryptophane and methionine(Met); The polar neutral amino acids comprises glycine, Serine, Threonine, halfcystine, tyrosine, asparagine and glutamine; (substantially) amino acid of positively charged comprises arginine, Methionin and Histidine; Electronegative (acid) amino acid comprises aspartic acid and L-glutamic acid.In addition, glycine and proline(Pro) are to influence the localized residue of chain.Non-conservative replacement requires the material with another kind of exchange of substance of one of these kinds.In addition, if necessary, the amino acid whose analogue of nonclassical amino acid or chemistry can be imported as substituent or adds in the antibody sequence.Non-classical amino acid comprises, but be not limited only to the D-isomer of common amino acid, α-An Jiyidingsuan, the 4-aminobutyric acid, Gamma Amino Butyric Acid, the 2-aminobutyric acid, γ-Gamma Amino Butyric Acid, epsilon-amino caproic acid, 6-aminocaprolc acid, α-An Jiyidingsuan, the 2-aminoisobutyric acid, the 3-alanine, ornithine, nor-leucine, norvaline, oxyproline, sarkosine, citrulline, cysteic acid, t-butyl glycine, t-butyl L-Ala, phenylglycine, Cyclohexylalanine, Beta-alanine, fluorescence-amino acid, the amino acid of artificial design is Beta-methyl amino acid for example, C Alpha-Methyl amino acid, N Alpha-Methyl amino acid and common amino acid analogue.

In another embodiment, the site that is used to change of selection is to reach avidity maturation (referring to above) with phage display.

Available any induced-mutation technique known in the art changes the single Nucleotide in the dna sequence dna, in order to prepare the aminoacid replacement thing in antibody sequence, or in order to create/remove restriction site to promote more mutagenesis.Such technology includes, but not limited to chemomorphosis, external site-directed mutagenesis (Kunkel, Proc.Natl.Acad.Sci.USA, 82:488 (1985); Hutchinson, people such as C., J.Biol.Chem.253:6551 (1978)), oligonucleotide-directed mutagenesis (Smith, Ann.Rev.Genet, 19:423-463 (1985); People such as Hill, Methods Enzymol, 155:558-568 (1987)), the overlapping expansion of PCR-based (people such as Ho, Gene, 77:51-59 (1989)), big primer mutagenesis of PCR-based (people such as Sarkar, Biotechniques, 8:404-407 (1990)) etc.The dideoxy dna order-checking of available two strands is determined to change.

In certain embodiments of the invention, can change anti-CD 19 antibodies and produce fusion rotein; Be antibody, or be fused to the fragment of foreign preteins, polypeptide or peptide.In certain embodiments, the albumen that is fused to the part of anti-CD 19 antibodies is the enzyme component of ADEPT.Can be used as other albumen of the albumen use of merging with anti-CD 19 antibodies or the example of polypeptide comprises, but be not limited only to, toxin for example ricin, toxalbumin, rnase, DNase I, staphylococcic enterotoxin-A, Pokeweed antiviral protein matter, spend more white tree inhibitor (gelonin), diphtheria toxin toxin, Rhodopseudomonas extracellular toxin and Rhodopseudomonas intracellular toxin.Referring to, for example: people such as Pastan, Cell, people such as 47:641 (1986) and Goldenberg, Cancer Journal forClinicians, 44:43 (1994).The active toxin of spendable enzymatic and fragment thereof comprise diphtheria A chain, the uncombined active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-Zhou Qujunsu, tung oil tree albumen, alizarin albumen, dyers' grapes albumen (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, the medicinal inhibitor of sapaonaria, spend more white tree inhibitor (gelonin), mitogellin, restrictocin, phenomycin, enomycin and tricothecenes.Referring to, for example: on October 28th, 1993 disclosed WO 93/21232.

Can reorganize by gene, the method for motif reorganization, exon reorganization and/or codon reorganization (being known as " DNA reorganization " jointly) produces additional fusion rotein.Available DNA reorganizes and changes SYNAGIS

Or the activity of its fragment (for example: tool is than antibody or its fragment of the high affinity and the low rate of dissociation).Referring to, in general, U.S. Patent number 5,605,793; 5,811,238; 5,830,721; 5,834,252 and 5,837,458, and people such as Patten, 1997, Curr.OpinionBiotechnol, 8:724-33; Harayama, 1998, Trends Biotechnol.16 (2): 76-82; People such as Hansson, 1999, J.Mol.Biol, 287:265-76; And Lorenzo and Blasco, 1998, Biotechniques 24 (2): 308-313 (described each patent and publication are incorporated into herein as a reference with its integral body).US publication 20030118592 as people such as Ledbetter, US publication 200330133939 and PCT publication number WO 02/056910 are described, antibody can further be the albumen that the binding domains immunoglobulin (Ig) merges, and described reference is incorporated into herein as a reference with its integral body.

5.1.9 domain antibodies

Anti-CD 19 antibodies in the compositions and methods of the invention can be a domain antibodies, and for example: comprise the antibody of little functional combining unit, it is corresponding to the variable region of human antibody heavy chain (VH) or light chain (VL).The example of domain antibodies include but not limited to, by Domantis company limited (Cambridge, UK) and Domantis company (Cambridge, MA USA) provide, be specific to the treatment target domain antibodies (referring to, for example WO 04/058821; WO 04/003019; U.S. Patent number 6,291,158; 6,582,915; 6,696,245 and 6,593,081).The antibody of anti-CD19 structural domain is discerned in available available commercially domain antibodies storehouse.In certain embodiments, anti-CD 19 antibodies of the present invention comprises a functional combining unit of CD19 and a Fc γ function of receptors combining unit.

5.1.10 double antibody

Term " double antibody " refers to have the little antibody fragment of two antigen binding sites, and this fragment is included in identical polypeptide chain (V _H-V _L) in light chain variable territory (V _L) continuous heavy chain variable domain (V _H).Be short to by use and do not allow paired connector between two structural domains on the same chain, structural domain is matched mutually with the complementary structure territory of another chain, and create two antigen binding sites.For being described in more comprehensively of complete body, for example EP 404,097; WO 93/11161; With people such as Hollinger, Proc.Natl.Acad.Sd.USA is among the 90:6444-6448 (1993).

5.1.11 vaccine body (vaccibody)

In certain embodiments of the invention, anti-CD 19 antibodies is the vaccine body.The vaccine body is the dimerization polypeptide.Each monomer of vaccine body is had specific scFv for the APC surface molecular and is formed by what be connected with second scFv by hinge region and C γ 3 structural domains.In other embodiments of the present invention, the vaccine body that comprises one of the anti-CD 19 antibodies fragment of scFv can be used for B cell that those are remained to be damaged and mediation ADCC the effector cell side by side.For example: referring to, people such as Bogen, U.S. Patent Application Publication No. 20040253238.

5.1.12 linear antibody

In certain embodiments of the invention, anti-CD 19 antibodies is linear antibody.Linear antibody comprises the Fd fragment (V of pair of series _H-C _H1-V _H-C _H1), they have formed a pair of antigen binding domain.That linear antibody can be dual specific or monospecific.Referring to, people such as Zapata, ProteinEng.8 (10): 1057-1062 (1995).

5.1.13 parental generation antibody

In certain embodiments of the invention, anti-CD 19 antibodies is a parental generation antibody." parental generation antibody " refers to that the antibody of the disclosed change in place therewith/sudden change compares, and is included in its one or more hypervariable regions or lacks or lack the antibody of the aminoacid sequence of one or more amino-acid residues near its place, one or more hypervariable region.Therefore, as disclosed here, parental generation antibody is compared with corresponding antibody mutant hypervariable region, has shorter hypervariable region.The parental generation polypeptide can comprise native sequences (promptly naturally occurring) antibody (comprising naturally occurring allelic variant) or be pre-existing in the natural antibody (for example other interpolation, disappearance and/or replacement) that has sequence that aminoacid sequence changes.The preferably humanized antibody of parental generation antibody or people's antibody.

5.1.14 antibody fragment

" antibody fragment " comprises the part of full length antibody, generally has its antigen combination or variable region.The example of antibody fragment comprises Fab, Fab ', the F (ab ') that is formed by antibody fragment ₂With the Fv fragment; Complete body; Linear antibody; Single-chain antibody molecule and multi-specificity antibody.

Traditionally, the soluble protein digestion via to complete antibody obtains these fragments.(referring to, for example: people such as Morimoto, Journal of Biochemical and Biophysical Methods, people such as 24:107-117 (1992) and Brennan, Science, 229:81 (1985)).But, can directly produce these fragments now by recombinant host cell.For example: can isolate antibody fragment in antibody phage library from above.Optionally, can be directly reclaim Fab '-SH fragment, and carry out chemistry and be connected to form F (ab ') from E.coli ₂Fragment (people such as Carter, Bio/Technology, 10:163-167 (1992)).According to another method, can from the recombinant host cell substratum, directly isolate F (ab ') ₂Fragment.Other technology that produces antibody fragment is conspicuous to skilled doctor.In other embodiment, the antibody of selection is strand Fv fragment (scFv).Referring to, for example: WO 93/16185.In certain embodiments, antibody is not the Fab fragment.

5.1.15 bi-specific antibody

Bi-specific antibody is the antibody that at least two different epi-positions is had binding specificity.Typical bi-specific antibody can be attached on two different epi-positions of B cell surface marker.Other such antibody can be in conjunction with first B cell marking, again in conjunction with second B cell surface marker.Optionally, anti-B cell marking brachium conjunctivum can with at white cell, the arm that for example combines trigger molecule on the Fc acceptor (Fc γ R) of TXi Baoshouti molecule (for example CD2 or CD3) or IgG combines, so that the defense mechanism of cell is concentrated on the B cell.Also available bi-specific antibody is positioned cytotoxin reagent on the B cell.These antibody have B cell marking brachium conjunctivum and in conjunction with cytotoxin (for example: saponaretin (saporin), anti-interferon- ^*, vincaleucoblastine, ricin A chain, methola-exate or radio isotope haptens) arm.(for example: F (ab '): bi-specific antibody) bi-specific antibody can be prepared into full length antibody or antibody fragment.

In the method for preparing bi-specific antibody known in the art.(referring to, for example: people such as Millstein, Nature, 305:537-539 (1983); People such as Traunecker, EMBO be (1991) J.10:3655-3659; People such as Suresh, Methods in Enzymology, 121:210 (1986); People such as Kostelny, J.Immunol, 148 (5): 1547-1553 (1992); People such as Hollinger, Proc.NatlAcad Sci.USA, 90:6444-6448 (1993); People such as Gruber, J.Immunol.152:5368 (1994); U.S. Patent number 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,81; 95,731,168; 4,676,980 and 4,676,980; WO 94/04690; WO 91/00360; WO 92/200373; WO 93/17715; WO 92/08802 and EP 03089.)

In certain embodiments of the invention, the CD3 ε chain that composition and method do not comprise people's CD19 and TXi Baoshouti has specific dual specific Muridae antibody, people such as Daniel for example, Blood, the described bi-specific antibody of 92:4750-4757 (1998).In preferred embodiments, anti-CD 19 antibodies in the compositions and methods of the invention is a bi-specific antibody, that anti-CD 19 antibodies is behaved or humanized, and has specificity for people CD19 and the epi-position on the T cell, maybe can be attached to people's effector cell, for example, monocyte/macrophage and/or influence the natural killer cell of necrocytosis.

5.1.16 the function of through engineering approaches effector

Can be according to the changing function of effector anti-CD 19 antibodies of the present invention, to strengthen the effect of antibody in treating B cell malignancies for example.For example: cysteine residues can be imported the Fc district, form the interchain disulfide linkage thereby make in this district.So the homotype dimerization antibody that produces can improve the internalization ability and/or increase the cell killing of complement-mediated and/or the cytotoxicity (ADCC) of antibody dependent cellular mediation.Referring to, people such as Caron, J.Exp Med.176:1191-1195 (1992) and Shopes, B.J.Immunol.148:2918-2922 (1992).Also people such as available Wolff, Cancer Research, the crosslinked homotype dimerization antibody for preparing of isodigeranyl function that 53:2560-2565 (1993) describes with enhanced anti-tumor activity.Optionally, antibody can be designed to have two Fc district, thereby strengthen the ability of complement dissolving and ADCC.Referring to, people such as Stevenson, Anti-Cancer Drug Design, 3:219-230 (1989).

In designerantibodies Fc known in the art district, so that other method of its change effector function (for example: people such as Koenig, U.S. Patent Publication No. 20040185045 and PCT publication number WO2004/016750, it has described change Fc district, thereby with the binding affinity of FC γ RIIA is compared, strengthened binding affinity to Fc γ RIIB; Also referring to, people such as Armour, PCT publication number WO 99/58572, people such as Idusogie, people such as WO 99/51642 and Deo, U.S.6,395,272; The content of described reference is incorporated into herein with its integral body).In change also known in the art Fc district with reduction for the method for the binding affinity of Fc γ RIIB (for example: people such as Ravetch, U.S. Patent Publication No. 20010036459 and PCT publication number WO 01/79299, described reference is incorporated into herein with its integral body).Also described with wild-type Fc district and compared, have for Fc γ RIIIA and/or Fc γ RIIA the enhanced binding affinity variant Fc district modified antibodies (for example: people such as Stavenhagen, PCT publication number WO 2004/063351, described reference is incorporated into herein with its integral body).

Whether available external check known in the art is determined at the anti-CD 19 antibodies that uses in the compositions and methods of the invention can regulate ADCC, as described in the 5.3.2 joint.

5.1.17 variant Fc district

The invention provides the proteic prescription in the Fc district that comprises change.Be the non-Fc district that exists naturally, for example comprise the Fc district of one or more non-amino-acid residues that exist naturally.The Fc district of change of the present invention also comprises the Fc district of containing aminoacid deletion, interpolation and/or change.

Known Fc district used herein comprises the polypeptide that contains the antibody constant region except that the first constant region immunoglobulin domains.Therefore Fc refers to preceding two constant region immunoglobulin domains of IgA, IgD and IgG, first three constant region immunoglobulin domains of IgE and IgM, and the deformable hinge N-end of these structural domains.For IgA and IgM, Fc can comprise the J chain.For IgG, Fc comprises the hinge between immunoglobulin domains Cgamma2 and Cgamma3 (C γ 2 and C γ 3) and Cgamma1 (C γ 1) and the Cgamma2 (C γ 2).Though the border in Fc district can change, but usually with human IgG heavy chain Fc area definition for to comprise residue C226 or P230 at its C-terminal, its number is according to people such as Kabat (1991, NIHPublication 91-3242, National Technical Information Service, Springfield, EU index VA)." Kabat propose EU index " refers to the residue number of the described human IgG1 EU of people antibody such as above-mentioned Kabat.Fc can refer to this district in isolating this district or antibody, antibody fragment or the Fc fusion rotein scope.The Fc misfolded proteins can be antibody, Fc syzygy or any albumen or the protein structure domain that comprises the Fc district.Especially preferably the albumen that comprises variant Fc district, it is the non-Fc variant that exists naturally.Attention: the number in the Fc site is observed polymorphism, and it includes but are not limited to Kabat270, and 272,312,315,356 and 358, therefore between these sequences and sequence of the prior art, may there be nuance.

The present invention includes the Fc variant proteins, it has altered binding characteristic with respect to comparable molecule (for example except having wild-type Fc district, having the albumen of same acid sequence) for Fc part (for example Fc acceptor, C1q).The example of binding characteristic include but not limited to, binding specificity, balance ionization constant (KD), decomposition and combination rate (being respectively Koff and Kon), binding affinity and/or activity.It is generally acknowledged, preferably to have the binding molecule of low KD with respect to the binding molecule with high KD (for example: Fc variant proteins, for example antibody).Yet, in that the value of kon or koff may be more relevant than the value of KD in some cases.Those skilled in the art can determine that it is topmost which kinetic parameter is used for given antibody.

The Fc district is for the avidity of its part with in conjunction with character, can known in the artly be used to measure the interactional external test method of Fc-Fc γ R (based on the test of biological chemistry or immunology) and measure by various, be that the Fc district combines with the specificity of Fc γ R, include but are not limited to, null readings (for example: enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA), or kinetics (for example: BIACORE ^RAnalyze) and other method is for example indirectly in conjunction with mensuration, competitive inhibition mensuration, fluorescence resonance energy transmission (FRET), gel electrophoresis and chromatography (for example: gel-filtration).These and other method can be used on the marker detection on one or more components and/or uses various detection methods, includes but are not limited to color development, fluorescence, luminescent or isotropic marker.Binding affinity and dynamic (dynamical) detailed description can be referring to Paul, W.E.ed.Fundamental Immunology, and 4th Ed.Lippincott-Raven, Philadelphia (1999), it concentrates on antibody-immunogen and interacts.

In one embodiment, with respect to comparable molecule, the Fc misfolded proteins improves the ability of closing one or more Fc parts of finishing.In another embodiment, the Fc misfolded proteins has with comparable molecule compares at least 2 times for the avidity of Fc part, or at least 3 times, or at least 5 times, or at least 7 times, or at least 10 times, or at least 20 times, or at least 30 times, or at least 40 times, or at least 50 times, or at least 60 times, or at least 70 times, or at least 80 times, or at least 90 times, or at least 100 times, or at least 200 times the avidity for the Fc part.In specific embodiment, the Fc misfolded proteins has improved the ability in conjunction with the Fc acceptor.In another specific embodiment, the Fc variant proteins has improved the ability in conjunction with Fc acceptor FcRn.In another specific embodiment, the Fc variant proteins has improved ability in conjunction with C1q with respect to comparable molecule.

Can prolong the proteic serum half life that comprises the Fc district to the binding affinity of FcRn by increasing the Fc district.In one embodiment, with respect to comparable molecule, prolonged the serum half life of Fc misfolded proteins.

" cytotoxicity of antibody dependent cellular mediation " or " ADCC " phalangeal cell toxicity make these cytotoxic effector cells be attached to antigen specifically and produce on the target cell, and finally killing target cell with cytotoxin, described cytotoxicity excretory Ig combines with the Fc acceptor (FcRs) that certain cytotoxin cell (for example: natural killer (NK) cell, neutrophil(e) cell and scavenger cell) presents.Specific high affinity IgG antibody points to the surface of the target cell of " equipment " cytotoxin cell, and such the killing and wounding of absolute demand.Target cell be dissolved in the extracellular, require to point to cell-, do not relate to complement to the contact of-cell.Can think, except that antibody, comprise other albumen in Fc district, have the Fc fusion rotein of specific combination in particular, will influence cell-mediated cytotoxicity to antigen generation target cell ability.In brief, the cell-mediated cytotoxicity that is caused by the activity of Fc fusion rotein also refers to ADCC activity herein.

Measured any specific Fc misfolded proteins mediates target cell by ADCC dissolved ability.In order to measure the ADCC activity, with the Fc misfolded proteins of research add with immune effector cell bonded target cell in, its available immune complex activates and causes the cytolysis of target cell.Check cytolysis roughly by from dissolved cell, removing mark (for example: radioactive substrates, fluorescence dye or natural intracellular protein).The useful effector cell of such check is comprised peripheral blood monocyte (PBMC) and natural killer (NK) cell.The object lesson of external ADCC check is people such as Wisecarver, and 1985,79:277-282; People such as Bruggemann, 1987, J Exp Med 166:1351-1361; People such as Wilkinson, 2001, J ImmunolMethods 258:183-191; People such as Patel, 1995 J Immunol Methods 184:29-38.Alternatively or replenish ground, the ADCC activity of the Fc misfolded proteins of research can be measured in vivo, for example: animal model people such as Clynes for example, 1998, in the disclosed animal model of PNAS USA 95:652-656.

In one embodiment, with respect to comparable molecule, the Fc variant proteins has enhanced ADCC activity.In one embodiment, the Fc misfolded proteins has the ADCC activity of comparing at least 2 times or at least 3 times or at least 5 times or at least 10 times or at least 50 times or at least 100 times with comparable molecule.In another specific embodiment, the Fc misfolded proteins has the enhanced binding ability for Fc acceptor Fc γ RIIIA with respect to comparable molecule, and has enhanced ADCC activity.In other embodiment, the Fc misfolded proteins has enhanced ADCC activity with respect to comparable molecule, and has the serum half life of prolongation.

" cytotoxicity of complement-dependent " and " CDC " refer to the cytolysis of target cell in the presence of complement.First component (C1q) by complement system is attached on the molecule, combines with isogeneic as antibody, comes initial complement activation approach.Measure complement activation, can implement CDC and detect, people such as Gazzano-Santoro for example, l996, J.Immunol.Methods, the description of 202:163.In one embodiment, with respect to comparable molecule, the Fc misfolded proteins has enhanced CDC activity.In one embodiment, the Fc misfolded proteins has the CDC activity of comparing at least 2 times or at least 3 times or at least 5 times or at least 10 times or at least 50 times or at least 100 times with comparable molecule.In other embodiments, with respect to comparable molecule, the Fc misfolded proteins has the serum half life that enhanced CDC is active and prolong.

In one embodiment, the invention provides a kind of formulation, wherein the Fc district be included in by the EU index number that proposes according to Kabat obtain 234,235,236,239,240,241,243,244,245,247,252,254,256,262,263,264,265,266,267,269,296,297,298,299,313,325,326,327, the amino-acid residue that non-natural exists on one or more sites of selecting in 328,329,330,332,333 and 334 groups of being formed.At random, the Fc district can comprise well known by persons skilled in the art, the amino-acid residue that the non-natural in interpolation and/or replacement site produces (referring to, for example: United States Patent (USP) 5,624,821; 6,277,375; 6,737,056; PCT patent disclosure text WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO 04/035752 and WO 05/040217).

In one embodiment, the invention provides the formulation of Fc misfolded proteins, wherein the Fc district is included in the 234D that is obtained by the EU index number that proposes according to Kabat, 234E, 234N, 234Q5,234T, 234H, 234Y, 2341,234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 2351,235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241R, 243W, 243L, 243Y, 243R, 243Q, 244H, 245A, 247V, 247G, 252Y, 254T, 256E, 262I, 262A, 262T, 262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 269H, 269Y, 269F, 269R, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 313F, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 3301,330F, 330R, 330H, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, the amino-acid residue that at least one non-natural of selecting in the group that 332Y and 332A formed exists.At random, the Fc district can comprise the amino-acid residue that non-natural well known by persons skilled in the art interpolation and/or that replace exists (referring to, for example: United States Patent (USP) 5,624,821; 6,277,375; 6,737,056; PCT patent disclosure text WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO 04/035752 and WO 05/040217).

In another embodiment, the invention provides the formulation of Fc variant proteins, wherein the Fc district is included in the amino-acid residue of at least one non-natural existence of selecting in 239,330 and 332 groups of being formed that obtained by the EU index number that proposes according to Kabat.In a specific embodiment, the invention provides a kind of formulation of Fc misfolded proteins, wherein the Fc district is included in the 239D that is obtained by the EU index number that proposes according to Kabat, the amino acid that non-natural exists on one or more sites of selecting in the group that 330L and 332E5 formed.Randomly, the Fc district can further be included in the amino acid that non-natural exists on one or more sites of selecting in 252,254 and 256 groups of being formed that obtained by the EU index number that proposes according to Kabat.In specific embodiment, the invention provides a kind of formulation of Fc misfolded proteins, wherein the Fc district is included in the 239D that is obtained by the EU index number that proposes according to Kabat, the amino acid that at least one non-natural of selecting in the group that 330L and 332E5 formed exists and at the 252Y that obtains by the EU index number that proposes according to Kabat, the amino acid that non-natural exists on one or more sites of selecting in the group that 254T and 256E formed.

In one embodiment, Fc variant of the present invention can combine with other known Fc variant, and described other Fc variant is for example people such as Ghetie, and 1997, Nat Biotech.15:637-40; People such as Duncan, 1988, Nature 332:563-564; People such as Lund, 1991, J.Immunol147:2657-2662; People such as Lund, 1992, Mol Immunol29:53-59; People such as Alegre, 1994, Transplantation 57:1537-1543; People such as Hutchins, 1995, Proc Natl.Acad Sci USA 92:11980-11984; People such as Jefferis, 1995, Immunol Lett.44:111-117; People such as Lund, 1995, Faseb is J.9:115-119; People such as Jefferis, 1996, Immunol Lett 54:101-104; People such as Lund, 1996, J Immunol 157:4963-4969; People such as Armour, 1999, Eur J Immunol 29:2613-2624; People such as Idusogie, 2000, J Immunol 164:4178-4184; People such as Reddy, 2000, J Immunol 164:1925-1933; People such as Xu, 2000, Cell Immunol 200:16-26; People such as Idusogie, 2001, J Immunol 166:2571-2575; People such as Shields, 2001, J BiolChem 276:659 1-6604; People such as Jefferis, 2002, Immunol Lett 82:57-65; People such as Presta, 2002, Biochem Soc Trans 30:487-490; U.S. Patent number 5,624,821; 5,885,573; 5,677,425; 6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260; 6,528,624; 6,194,551; 6,737,056; 6,821,505; 6,277,375; The open text WO 94/29351 of U.S. Patent Publication No. 2004/0002587 and PCT; WO99/58572; WO 00/42072; WO 02/060919; WO 04/029207; WO 04/099249; Disclosed Fc variant among the WO 04/063351.The present invention also comprises the Fc district of containing disappearance, adding and/or change.Apparent to those skilled in the art to other change/replacement/interpolation/disappearance that the Fc district carries out.

Be known in the art the method in the Fc district that produces the non-natural existence.For example: can produce amino acid whose replacement and/or disappearance by mutafacient system, described mutafacient system includes but are not limited to, site-directed mutagenesis (Kunkel, Proc.Natl.Acad.Sci.USA 82:488-492 (1985)), PCR mutagenesis (Higuchi, at " PCR Protocols:AGuide to Methods andApplications ", Academic Press, San Diego, among the pp.177-183 (1990)) and cassette mutagenesis (people such as Wells, Gene 34:315-323 (1 985)).Preferably, with overlapping-the expansion PCR method is implemented site-directed mutagenesis, and (Higuchi is at " PCR Technology:Principlesand Applications for DNA Amplification ", Stockton Press, New York is among the pp.61-70 (1989)).Optionally, available overlapping-expansion round pcr (Higuchi, the sudden change importing target sequence of ibid) will be any wanting (initiate dna).For example: the first round PCR of overlapping-extended method comprises with outside primer (primer 1) and inner mutagenic primer (primer 3) amplified target sequence, use second outside primer (primer 4) and inner primer (primer 2) respectively, obtain two PCR fragments (Segment A and B).Inner mutagenic primer (primer 3) is designed to comprise and specifies the mispairing want the target sequence that suddenlys change.Take turns among the PCR second, with the product (Segment A and B) of two outside primers (primer 1 and 4) by pcr amplification first round PCR.Digest the PCR full length fragment (fragment C) that produces with restriction enzyme, and the restriction fragment that produces is cloned in the appropriate carriers.As the first step of mutagenesis, initiate dna (for example: coding Fc fusion rotein, antibody or Fc district only) can be cloned in the mutagenesis carrier with implementing.Introduction is designed to reflect the aminoacid replacement of wanting.Be known in the art produce other useful method of variant Fc district (referring to, for example: U.S. Patent number 5,624,821; 5,885,573; 5,677,425; 6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260; 6,528,624; 6,194,551; 6,737,056; 6,821,505; 6,277,375; The open text WO 94/29351 of U.S. Patent Publication No. 2004/0002587 and PCT; WO99/58572; WO 00/42072; WO 02/060919; WO 04/029207; WO 04/099249; WO 04/063351).

In certain embodiments, the Fc misfolded proteins comprises the sugared shape of one or more through engineering approaches, promptly is covalently attached to the carbohydrate ingredient of the molecule that comprises the Fc district.The sugared shape of through engineering approaches can be used for various uses, includes but are not limited to, and strengthens or reduce the function of effector.The sugared shape of available any method production engineeringization well known by persons skilled in the art, for example by bacterial strain through engineering approaches or that variant is expressed, by with the coexpression of one or more enzymes, for example DIN-acetylglucosaminyltrVnsferase III (GnTI11), by being expressed in various organisms or from the molecule that comprises the Fc district in the various organic clones, or after the developed by molecule that comprises the Fc district by changing carbohydrate.Be known in the art the method for the sugared shape that produces through engineering approaches, include but are not limited to people such as Umana, 1999, Nat.Biotechnol 17:176-180; People such as Davies, 20017Biotechnol Bioeng 74:288-294; People such as Shields, 2002, J Biol Chem 277:26733-26740; People such as Shinkawa, 2003, J Biol Chem 278:3466-3473) U.S. Patent number 6,602,684; U.S.Ser.No.10/277,370; U.S.Ser.No.10/113,929; PCT WO 00/61739A1; PCT WO 01/292246A1; PCT WO 02/311140A1; PCT WO 02/30954A 1; Potillegent ^TMTechnology (Biowa, Inc.Princeton, N.J.); GlycoMAb ^TMGlycosylation engineering technology (GLYCARTbiotechnology AG, Zurich, Switzerland) method described in.Referring to, for example: WO 00061739; EA 01229125; US 20030115614; People such as Okazaki, 2004, JMB, 336:1239-49.

5.1.18 the glycosylation of antibody

In another embodiment, changed antibody glycosylation used according to the present invention.For example: can prepare glycosylated antibody (promptly lacking glycosylated antibody).Can change glycosylation extremely, for example: increase the avidity of antibody to target antigen.Such carbohydrate changes and can be attended by, for example: in the glycosylated one or more sites of the inner change of antibody sequence.For example: prepare one or more aminoacid replacement things, cause the elimination of one or more variable regions framework glycosylation site, thereby remove the glycosylation on this site.Such glycosylation can increase antibody to antigenic avidity.At U.S. Patent number 5,714, this method has been described in more detail in 350 and 6,350,861.Optionally, can prepare and cause that Fc district glycosylation site (for example: one or more aminoacid replacement things of the Xiao Chuing asparagine 297 of IgG).In addition, can in the bacterial cell that lacks necessary glycosylation structure, prepare glycosylated antibody.

In addition or optionally, can prepare antibody, the hypofluorescence antibody that the amount that for example has a fluorescent residue has reduced or have the antibody that has increased bifurcated GIcNAc structure with glycosylation variant.The glycosylation pattern of Gai Bianing has been proved to be the ADCC ability that can strengthen antibody like this.Such carbohydrate changes and can be attended by, for example: have expressing antibodies in the host cell of glycosylation structure of change.Cell described in the art with glycosylation structure of change, thus and can produce and have the host cell that changes glycosylated antibody used as expressing recombinant antibody of the present invention.Referring to, for example: Shields, people such as R.L. (2002) J.Biol.Chem.277:26733-26740; People such as Umana (1999) Nat.Biotech.17:176-1, and european patent number EP1,176,195; The open text WO 03/035835 of PCT; WO 99/54342.

5.2 the mass preparation/production of anti-CD 19 antibodies

In case the anti-CD 19 antibodies that will want has designed, with regard to the method for available extensive Antibody Preparation known in the art with the industrial-scale production anti-CD 19 antibodies.For example: available recombinant expression system is finished production, and described recombinant expression system for example but be not limited only to system as described below.

5.2.1 recombinant expression system

Antibody of the present invention or its variant recombinant expressed requires to make up the expression vector of the polynucleotide that comprise encoding antibody usually.In case obtain the polynucleotide of encoding antibody molecule of the present invention or heavy chain of antibody or light chain or its part (preferred, but unnecessary, as to comprise the variable region of heavy chain or light chain), available recombinant DNA technology known in the art produces the carrier that is used for preparing antibody molecule.Referring to, for example: U.S. Patent number 6,331,415, described reference is incorporated into herein as a reference with its integral body.Therefore, describe the polynucleotide that comprise the antibody coding nucleotide sequence by expression herein and prepared proteic method.Available method well-known to those skilled in the art makes up and comprises antibody coding sequence and the suitable expression vector of transcribing and translate control signal.These methods comprise that for example: extracorporeal recombinant DNA technology, synthetic technology and vivo gene are recombinated.Therefore, the invention provides and comprise heavy chain or the variable region of light chain that can operate the coding that links to each other antibody molecule of the present invention, heavy chain of antibody or light chain, antibody or its part with promotor, or the reproducible carrier of the nucleotide sequence of heavy chain or light chain CDR.Such carrier can comprise encoding antibody molecule constant region nucleotide sequence (referring to, for example: international publication number WO 86/05807 and WO 89/01036; And antibody variable region can be cloned in the carrier of expressing whole piece heavy chain, whole piece light chain or whole piece heavy chain and light chain and U.S. Patent number 5,122,464).

In embodiment optionally, the target homology mixture of all or part of anti-CD 19 antibodies of available generation prepare the compositions and methods of the invention anti-CD 19 antibodies (referring to, U.S. Patent number 6,063,630,6,187,305 and 6,692,737).In certain embodiments, the recombinant technology at random of all or part of anti-CD 19 antibodies of available production prepare the anti-CD 19 antibodies of the compositions and methods of the invention (referring to, U.S. Patent number 6,361,972,6,524,818,6,541,221 and 6,623,958).Also can use the locus specificity homologous recombination of Cre-mediation, in the cell of the antibody of the genome sequence of the cell of the immunoglobulin locus of expressing self-contained change, produce anti-CD 19 antibodies (referring to, U.S. Patent number 6,091,001).When wanting to produce people's antibody, host cell should be human cell system.These methods can be advantageously used in design the stable clone of permanent expressed antibody molecule.

In case with ordinary method expression vector is transformed in the host cell, just cultivates transformant then with the routine techniques of production antibody of the present invention.Therefore, the present invention includes to contain and can operate the coding that links to each other antibody of the present invention or its fragment with promotor, or its heavy chain or light chain, or its part, or the host cell of the polynucleotide of single-chain antibody of the present invention.In expressing the embodiment preferred of double-stranded antibody, can be in the host cell of expressing whole immunoglobulin molecules the carrier of coexpression encoding heavy chain and light chain, details are as follows.

Available various host expresses carrier system express anti-CD 19 antibodies of the present invention that the preparation that can be used for anti-CD 19 antibodies produces or its part (referring to, for example: U.S. Patent number 5,807,715).For example: mammalian cell, as Chinese hamster ovary cell (CHO), with as be connected from the carrier of the main mediation early gene promoter sub-element of human cytomegalic inclusion disease virus, this is the effective expression system (people such as Foecking of antibody, Gene, people such as 45:101 (1986) and Cockett, Bio/Technology, 8:2 (1990)).In addition, the antibody sequence that can select to adjust insertion is expressed, or changes and process the host cell strain of antibody gene product with the ad hoc fashion of wanting.This modification of protein product (for example glycosylation) and processing (for example division) may be important concerning proteic function.Different host cells for the translation of albumen and gene product before processing and modify and to have typical case and special mechanism.Can select suitable clone or host system, guarantee for the antibody of expressing or the correct modification and the processing of its part.For this reason, can use and have the cyto-architectural eukaryotic host cell that main transcription product, glycosylation and the phosphorylation of gene product are suitably processed.Such mammalian host cell include but not limited to CHO, VERY, BHK, HeIa, COS, MDCK, 293,3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO (the rat bone marrow tumour cell system of any immunoglobulin chain of not endogenous generation) CRL7O3O and HsS78Bst cell.

In preferred embodiments, the available human cell who is come by the development of immortal human quasi-lymphocyte is that reorganization produces monoclonal people's anti-CD 19 antibodies.In preferred embodiments, available human cell is that PER.C6. (Crucell, Holland) reorganization produces monoclonal people's anti-CD 19 antibodies.

In the bacterium system, can select many expression vectors easily according to the purposes of the antibody molecule of expressing.For example: when wanting this antibody of mass production, may need to make the carrier of the fusion protein product high level expression that is easy to purifying.Such carrier include but not limited to, E.coli expression vector pUR278 (people such as Ruther, EMBO, 12:1791 (1983)), and wherein antibody coding sequence can be connected in the carrier in the framework of lacZ coding region separately, thereby has produced fusion rotein; PIN carrier (Inouye﹠amp; Inouye, 1985, Nucleic Acids Res.13:3101-3109 (1985); Van Heeke﹠amp; Schuster, 1989, J.Biol.Chem.24:5503-5509 (1989)) etc.Also available pGEX carrier comes to express allogenic polypeptide with the form of the fusion rotein of gsh 5-transferring enzyme (GST).Usually, such fusion rotein is soluble, and can be after with free glutathione wash-out, by absorption be incorporated into matrix gsh agar beads and come from dissolved cell easily that purifying comes out.The pGEX carrier design is become to comprise the cracking site of zymoplasm or factor Xa proteolytic enzyme, so that from the GST part, discharge clone's target gene product.

In the insect system, the carrier of Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus (AcNPV) as expression alien gene.Virus is grown in the greedy frugiperda cell in meadow.The nonessential region that antibody coding sequence can be cloned into separately virus (for example: polyhedron gene), and (for example: the polyhedrin promotor) place under the control of AcNPV promotor.

In mammalian host cell, can utilize many expression systems based on virus.If as expression vector, the antibody coding sequence of research can be connected in the control mixture that adenovirus is transcribed/translated to adenovirus, for example: late promoter and three leaders.Can this mosaic gene be inserted the adenoviral gene group by reorganization in external or the body then.The insertion of virus genomic nonessential region (for example E1 or E3 district) will produce can and can be in infected host expressed antibody molecule recombinant virus (for example: referring to, Logan﹠amp; Shenk, Proc.Natl.Acad.Sci.USA, 81:355-359 (1984)).Effective translation of the antibody coding sequence that inserts also may need special start signal.These signals comprise ATG initiator codon and catenation sequence.In addition, the common reading frame homophase of initiator codon and the encoding sequence of wanting is to guarantee the segmental translation of whole insertion.The translation control signal and the initiator codon of these external sources can have various sources, and natural all can with artificial.Can strengthen element, transcription terminator etc. by comprising suitable transcribing, improve expression efficient (referring to, for example: people such as Bittner, Methods in Enzymol, 153:51-544 (1987)).

For long-term, high yield ground produce recombinant protein, preferably stably express.For example: but the clone of design stability expressed antibody molecule.Than better with the transient expression system of the copy expression vector that comprises the viral initial point that duplicates, available suitable expression controlling elements (for example: promotor, enhanser, sequence, transcription terminator, polyadenylic acid site etc.) and optional mark are controlled the DNA transformed host cell.By importing DNA, engineering cell was grown in enrichment medium 1-2 days, it is transferred to select in the substratum then.Selected marker in recombinant plasmid has resistance to selecting, and allow cytotostatic ground with plasmid integration to its karyomit(e), and grow into focus, it can be cloned successively and increase is clone.The plasmid of available code anti-CD 19 antibodies imports gene/cDNA any clone that is adapted at producing in the substratum.Optionally, the plasmid of available being called " targeting vector " will be expressed controlling elements (for example: promotor, enhanser etc.) and be imported suitable chromosomal foci in the host cell, with the native gene of " activation " anti-CD 19 antibodies.

Available many selective systems include but are not limited to, herpes simplex virus thymidine kinase (people such as Wigler, Cell, 11:223 (1977)), xanthoglobulin horse purine phosphoribosyl transferase (Szybalska﹠amp; Szybalski, Proc.Natl.Acad.ScL USA, 48:202 (1992)) and adenine phosphoribosyl transferase (people such as Lowy, Cell, 22:8-17 (1980)) gene, can be respectively at tk ^-, hgprt ^-Or aprT ^-Use in the cell.Also the metabolic antagonist resistance can be used as the basis of selecting following gene: have dhfr to the methotrexate resistance (people such as Wigler, Natl.Acad.Sci.USA, 77:357 (1980); People such as O ' Hare, Proc.Natl.Acad.Sci.USA, 78:1527 (1981)); Gpt (the Mulligan﹠amp that mycophenolic acid is had resistance; Berg, Proc.Natl.Acad.Sci.USA, 78:2072 (1981)); Aminoglycoside G-418 there are resistance neo (Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann.Rev.Pharmacol.Toxicol.32:573-596 (1993); Mulligan, Science 260:926-932 (1993) and Morgan and Anderson, Ann.Rev.Biochem.62:191-217 (1993); 155-215 (1993)) and the hygro (people such as Santerre, Gene, 30:147 (1984)) that Totomycin is had resistance May, TIB TECH 11 (5):.Can be with common known method is applied to select the recombinant clone of wanting routinely in the recombinant DNA technology field, and description to this method, for example: people such as Ausubel. (eds.) CurrentProtocols in Molecular Biology, John Wiley﹠amp; Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); And at the 12nd and 13 chapters, people such as Dracopoli (eds.), Current Protocolsin Human Genetics, John Wiley﹠amp; Sons, NY (1994); People such as Colberre-Garapin, 1981, J.Mol.Biol, 150:1, described reference is incorporated into herein as a reference with its integral body.

Can by carrier increase improve antibody molecule expression level (for summary, referring to: Bebbington and Hentschel, The use of vectors based on gene amplification forthe expression of cloned genes in mammalian cells in DNA cloning, Vol.3.Academic Press, New York (1987)).When the mark in the carrier system of expressing antibodies be can increase the time, the increase of inhibitor level will increase the number of marker gene copy in the host cell substratum.Because amplification region links to each other with antibody gene, production of antibodies also will increase (people such as Grouse, Mol.Cell.Biol, 3:257 (1983)).Can comprise around chromatin remodeling technology and the transgene expression that improves with the form of activation manual transcription structural domain by using the recombination method known to the skilled and the instrument in recombinant protein production field, improve the expression level of antibody.

Available two expression vector cotransfection host cells of the present invention, first vector encoded are from the heavy chain of polypeptide, and second vector encoded is from the light chain of polypeptide.Two carriers can comprise same selected marker, and this mark is the heavy chain and the light chain of express polypeptide coequally.Optionally, can use coding and the heavy chain of energy express polypeptide and the single carrier of light chain.On these sites, light chain is placed on heavy chain before to avoid excessive deleterious free heavy chain (Proudfoot, Nature 322:562-65 (1986) and Kohler, 1980, Proc.Natl Acad.ScL USA, 77:2197 (1980)).The encoding sequence of heavy chain and light chain can comprise cDNA or genomic dna.

When with recombinant expressed generation antibody molecule of the present invention, in the available immunoglobulin molecules purification field known any method purifying it, for example: with chromatography (for example: ion-exchange, affinity, especially to the affinity and the coating column chromatography of the specific antigens after the albumin A), centrifugation, differential solubility or with the standard technique of any other purification of protein.In addition, antibody of the present invention or its fragment can be fused to other xenogenesis peptide sequence that promotion described herein or known in the art is purified.

5.2.2 the purification of antibody with separate

When using recombinant technology, can in intracellular cytosolic space, produce antibody, or direct secretion is in substratum.If in cell, produce antibody, as the first step, remove granular debris, comprise host cell or cytolysis fragment, for example: by centrifugation or ultrafiltration process.People such as Carter, Bio/Technology, 10:163-167 (1992) has described the step that is secreted into the antibody in the E.coli cytosolic space.Briefly, dissolved cell was stuck with paste above 30 minutes under the condition that sodium-acetate (pH3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) exist.Available centrifugation is removed cell debris.When the antibody mutation body is secreted in the substratum, will at first use commercial available albumen thickening filtration device from the supernatant liquor of this expression system usually, for example: Amicon or Millipore Pellicon ultra filtration unit.Any above-mentioned steps all can be used proteinase inhibitor, and for example PMSF comes the arrestin hydrolytic action, and can use the antibiotic growth that usually suppresses external contaminant.

Available for example hydroxyapatite chromatography method, hydrophobic interaction chromatography, ion exchange chromatography, gel electrophoresis, saturating dialysis and/or affinity chromatography or make jointly separately or with other purification step to be used for the antibody compositions that purifying prepares from cell.A-protein depends on kind and the isotype that is present in any immunoglobulin fc region in the antibody variants as the suitability of affinity ligands.Can use a-protein to come the antibody (people such as Lindmark, J.Immunol.Methods, 62:1-13 (1983)) of purifying based on people γ 1, γ 2 or γ 4 heavy chains.Recommendation is used protein Gs people such as (, EMBO is (1986) J.5:1567-1575) Guss to all mouse isotypes and people γ 3.The matrix that is attached with affinity ligands is agarose normally, but also can use other matrix.On the mechanics stable matrix for example controlled pore glass or many (vinylbenzene divinyl) benzene compare with agarose and can accelerate flow velocity, shorten the treatment time.For the antibody that comprises the CH3 district, can (J.T.Baker, Phillipsburg NJ) be used for purifying with Bakerbond ABX resin.According to the antibody that will reclaim, other technology that also can use protein purification is ion exchange column method, ethanol precipitation, RPHPLC (reversed-phase high-performance liquid chromatography) method (reversed-phase HPLC), silica chromatography, heparin chromatography, negatively charged ion or Zeo-karb sepharose chromatography (for example many aspartic acids post method), chromatofocusing method, SDS-PAGE method and ammonium sulfate precipitation method for example.

After any rough purification step, elution buffer between the about 2.5-4.5 of available pH, preferably operation (for example: from about 0-0.25M salt) under low salt concn comprises the antibody of research and the mixture of pollutent with low pH hydrophobic interaction chromatography processing.

5.3 curative anti-CD 19 antibodies

The anti-CD 19 antibodies that uses in the compositions and methods of the invention, preferred people's antibody or humanized antibody preferably mediate people's antibody or the humanized antibody of human ADCC, or select from known anti-CD 19 antibodies, preferably mediate the antibody of human ADCC.In certain embodiments, anti-CD 19 antibodies can be chimeric antibody.In certain embodiments, anti-CD 19 antibodies can be monoclonal people, humanized or chimeric anti-CD 19 antibodies.Be used for the preferred IgG1 of anti-CD 19 antibodies of the compositions and methods of the invention or the people's antibody or the humanized antibody of IgG3 people's isotype.In other embodiments, be used for the preferred IgG2 of anti-CD 19 antibodies of the compositions and methods of the invention or the antibody or the humanized antibody of IgG4 people's isotype, it preferably mediates antibody or the humanized antibody of ADCC.

When producing this antibody with aforesaid method, in other embodiments of the present invention, it is chimeric, humanized or make people's antibody that murine antibody HB12a described herein and HB12b or other commercial available anti-CD 19 antibodies can be.

For example: spendable known anti-CD 19 antibodies include but not limited to, HD37 (IgG1) (DAKO, Carpinteria, CA), BU12 (G.D.Johnson, University ofBirmingham, Birmingham, United Kingdom), 4G7 (IgG1) (Becton-Dickinson, Heidelberg, Germany), J4.119 (Beckman Coulter, Krefeld, Germany), B43 (PharMingen, San Diego, CA), SJ25C1 (BDPharMingen, San Diego, CA), FMC63 (IgG2a) (Chemicon Int ' l.Temecula, CA) (people such as Nicholson, Mol.Immunol, 34:1157-1165 (1997); People such as Pietersz, Cancer Immunol.Immunotherapy, people such as 41:53-60 (1995) and Zola, Immunol Cell Biol, 69:411-422 (1991)), B4 (IgG1) (Beckman Coulter, Miami, FL) people such as Nadler, J.Immunol, 131:244-250 (1983) and/or HD237 (IgG2b) (Fourth International Workshop on HumanLeukocyte Differentiation Antigens, Vienna, Austria, 1989 and people such as Pezzutto, J.Immunol, 138:2793-2799 (1987)).

In certain embodiments, anti-CD 19 antibodies of the present invention comprises the HB12a heavy chain (Fig. 5 A) of the aminoacid sequence that contains SEQ ID NO:2.In other embodiments, anti-CD 19 antibodies of the present invention comprises the HB12b heavy chain (Fig. 5 B) of the aminoacid sequence that contains SEQ ID NO:4.

In certain embodiments, anti-CD 19 antibodies of the present invention comprises the HB12a light chain (Fig. 6 A) of the aminoacid sequence that contains SEQ ID NO:16.In other embodiments, anti-CD 19 antibodies of the present invention comprises the HB12b light chain (Fig. 6 B) of the aminoacid sequence that contains SEQ ID NO:18.

In certain embodiments, antibody is that the isotype of known antibodies (for example: antibody for example mentioned above (for example: HB12a or HB12b) people's isotype of IgG1 or IgG3) can be regulated variant.

The anti-CD 19 antibodies that is used for the compositions and methods of the invention can be naked antibody, immune conjugate or fusion rotein.The above-mentioned anti-CD 19 antibodies that is used for the compositions and methods of the invention preferably can reduce or the patient's of its treatment of consumptive use B cell and circulation immunity sphaeroprotein.The B cell that consumes can be in round-robin B cell or in the particular tissues, for example but be not limited only to marrow, spleen, gut associated lymphatic tissue and/or lymphoglandula.Can by various mechanism for example antibody dependent cellular mediation cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC), B cell proliferation suppresses and/or the B necrocytosis induces (for example: pass through apoptosis) to realize this consumption." consumption " B cell means the minimizing of B cell in circulation B cell and/or the particular organization, is reduced by at least about 25%, 40%, 50%, 65%, 75%, 80%, 85%, 90%, 95% or more, as described in the 5.4.3 joint.In specific embodiment, in fact all detectable B cells have all consumed from circulation.

5.3.1 the screening of people's CD19 binding antibody

Available in conjunction with testing evaluation in conjunction with the antigenic antibody of people CD19.Available direct combination test or competition are carried out combination in conjunction with test and are tested.Available standards ELISA or standard flow spectrophotometry are tested and are detected combination.In directly in conjunction with test, test candidate antibody combines with people CD19 is antigenic.In certain embodiments, shaker test comprises, and in second step, measures the necrocytosis or the apoptotic ability of the B cell that causes expressing human CD19.On the other hand, competition combines the ability of the compound competition of people CD19 in conjunction with test determination candidate antibody with known anti-CD 19 antibodies or other.

In directly in conjunction with test, allowing under candidate's antibody and the people CD19 antigen bonded situation people CD19 antigen to be contacted with candidate's antibody.In conjunction with occurring in the solution or on the solid surface.Preferably, mark candidate antibody is used for detecting before this.Any detectable compound all can be used for mark, for example but be not limited only to, and luminescent, fluorescence or radio isotope or comprise same material or nonisotopically labelled group, for example enzyme or stain.Cultivating enough generation bonded after one period, the condition and the processing of reactant being removed antibody excessive or non-specific binding.Usually, this comprises with suitable buffer reagent flushing.At last, detect the existence of CD19 antibody complex.

In CBA, measure the inhibition of candidate's antibody or shift known anti-CD 19 antibodies (or other compound) and people CD19 antigen bonded ability.The known binding substances of the CD19 of mark can be mixed mutually with candidate's antibody, and place and do not have the adding of candidate's antibody, and under the condition that the interaction between them can normally take place.Can candidate antibody exist or situation about lacking under the amount of the CD19 binding substances of the known people of the combining CD19 of mark relatively.

In preferred embodiments, for the formation and the detection of enhancing antibody antigenic compound, one or more compositions can be fixed on solid surface and carry out the combination test.In various embodiments, solid support can be, but is not limited only to, polycarbonate, polystyrene, polypropylene, polyethylene, glass, nitrocotton, dextran, nylon, polyacrylamide and agarose.Underwork can comprise pearl, film, particulate, and the internal surface of reaction vessel is microtiter plates, test tube or other reaction vessel for example.Can realize the fixing of people CD19 or other composition by covalency or non-covalent adhering to.In one embodiment, people's CD19 antigen and negative contrast are connected in epi-position, glutathione S-transferase (GST) for example is so that with for example anti-GST of commercial available antibody (Santa Cruz Biotechnology) mediation adhering to solid surface.

For example, the available people CD19 antigen that is fixed on the solid support carries out this affinity in conjunction with test.Usually, the non-motion component with association reaction refers to candidate's anti-CD 19 antibodies herein, carries out mark and realizes detecting.Can use various marking methods, for example luminescent, chromophoric group, fluorescence or radio isotope or or comprise same material or nonisotopically labelled group, for example enzyme or stain.In preferred embodiments, candidate's anti-CD 19 antibodies can be used fluorophor, and for example (fluorescein isothiocyanate provides the Chemicals from Sigma to fluorescein isothiocyanate, St.Louis) comes mark.

At last, available any detection method known in the art detects the mark that remains in solid surface.For example: if with fluorophor mark candidate anti-CD 19 antibodies, available photofluorometer detects mixture.

Preferably,, or comprise the form of the antigenic separatory membrane of people CD19, people CD19 antigen is joined in conjunction with in the test with the antigenic intact cell of expressing human CD19.Therefore, can in intact cell of cultivating or animal model, under the situation that is with or without the existence of candidate's anti-CD 19 antibodies, detect antigenic direct combination to people CD19.Can be with the candidate's anti-CD 19 antibodies and the antigenic cell of expressing human CD19 of mark, or obtain to mix mutually from the crude extract of these cells, and can add candidate's anti-CD 19 antibodies.Available isolating film is identified and people CD19 bonded candidate anti-CD 19 antibodies.For example: in the model experiment that uses isolating film, available genetic engineering makes cell expressing people CD19 antigen.The available standards technology obtains film, and is used for external in conjunction with test.With candidate's anti-CD 19 antibodies of mark (for example: fluorescent-labeled antibody) be incorporated into film, and measure specific activity; Measure the specificity combination by comparing with the combination test under the situation about existing at excessive unmarked (cold) candidate anti-CD 19 antibodies.Optionally, soluble people CD19 antigen can be by recombinant expressed, and utilize the antibody that combines with people CD19 antigen to identify in based on acellular test.Can in based on acellular shaker test, use recombinant expressed people CD19 polypeptide.Optionally, can in based on acellular pilot system, use with one or more people CD19 antigen-binding portion thereof or comprise the corresponding peptide of fusion rotein of one or more people CD19 antigen-binding portion thereof, be incorporated into the antibody of people CD19 antigen part with evaluation.In based on acellular test, use methods known in the art that recombinant expressed people CD19 is attached to solid substrate for example on test tube, microtiter well or the post (referring to, people such as Ausubel, supra).The antigenic ability of determination test antibodies people CD19 then.

Optionally, can in solution, carry out association reaction.In this test, the component of permission mark combines with the binding partner in its solution.If the difference of size can allow to carry out this separation between the component of mark and its binding partner, then can be by the product of association reaction is realized separating by ultra-fine filter, the hole of described ultra-fine filter allows not the component of bonding mark to pass through, but does not allow its binding partner or the marker components that is incorporated on its binding partner is passed through.Also available any can be from solution the reagent of capture of labels component binding partner realize separating, described reagent for example: for the antibody of binding partner etc.

For example, in one embodiment, can with from the phage in the continuous phage display library by with solid phase for example the people CD19 antigen that comprises purifying that links to each other of plastic bead or the post of derivative, similar body, fragment or its structural domain, screen phage library.By changing the intensity of wash buffer, can increase expression has the peptide of high affinity to people CD19 antigen phage.Can clone isolating phage from the post, and directly measure its affinity.Know which antibody and its aminoacid sequence are the strongest to the combination of people CD19 antigen, the available computers model is identified the molecule contact between CD19 antigen and the candidate's antibody.

In another specific embodiment of this respect of the present invention, solid support is to comprise the antigenic film of people CD19 that is attached to microtiter plates.For example, candidate's antibody can combine with the cell of expression library antibody under the condition that allows the library member to express in microtiter plates.Results and people CD19 bonded library member.This method, usually in the mode of embodiment at Parmley and Smith, 1988, Gene, 73:305-318; People such as Fowlkes, 1992, BioTechniques, 13:422-427; PCT publication number WO94/18318 and description to some extent in the reference of above quoting.The antibody of identifying that combines with people CD19 antigen can be the antibody of above-described any kind or modified forms.

5.3.2 the screening of the people ADCC effector function of antibody

Preferably use the human IgG antibody-like in the present invention, because it has the functional performance of long half life in serum, can mediate the function (Monoclonal Antibodies:Principles and Applications, Wiley-Liss, Inc. the 1st chapter (1995)) of various effectors.The human IgG antibody-like can be further divided into following 4 subclass: IgG1, IgG2, IgG3 and IgG4.A lot of researchs have been carried out so far to ADCC and CDC with as the apoptosis activity of the effector function of IgG antibody-like, and reported in the antibody of human IgG class, the IgG1 subclass has the active and CDC activity (ChemicalImmunology of the highest ADCC in human body, 65,88 (1997)).

What active and CDC activity of the ADCC of human IgG1's subclass antibody and the active performance of apoptosis generally included antibody Fc district and the antibody receptor that is present in the effector cell surface combines (hereinafter referred to as " Fc γ R "), described effector cell such as killer cell, natural killer cell or activated macrophage.Can be in conjunction with various complement components.About combination, known to the hinge region of Antibody C region (hereinafter referred to as " C γ 2 districts ") and several amino acid residue very important (Eur.J.Immunol.23,1098 (1993), the Immunology of second structural domain, 86,319 (1995), ChemicalImmunology, 65,88 (1997)), and the sugar chain in C γ 2 districts (ChemicalImmunology, 65,88 (1997)) is also very important.

Consider effector function, can modify anti-CD 19 antibodies of the present invention, for example: the ADCC and/or complement dependent cytotoxicity (CDC) and/or the apoptosis activity that strengthen antibody.This can introduce one or more aminoacid replacement and realize by the Fc district at antibody.Optionally or replenish ground, can introduce cysteine residues in Fc district, make formation interchain disulfide bond in this district.Can produce homotype dimerization antibody like this, it has the cell killing and the ADCC (people such as Caron of the complement-mediated of the internalization ability of improvement and/or raising, J.Exp.Med.176:1191-1195 (1992) and Shopes, J Immunol.148:2918-2922 (1992)).The cross connection of also available isodigeranyl function produces the homotype dimerization antibody (people such as Wolff, Cancer Research, 53:2560-2565 (1993)) with enhanced anti-tumor activity.Also can be designed to have two or more Fc districts to antibody, cause enhancing people such as (, Anti-Cancer Drug Design, (3) 219-230 (1989)) Stevenson of complement dissolving and ADCC ability.

In designerantibodies Fc district known in the art so as other method that changes effector function (for example: people such as Koenig, U.S. Patent Publication No. 20040185045 and PCT publication number WO2004/016750, its described change the Fc district in case with the binding affinity of FC γ RIIA is compared the binding affinity of enhancing to Fc γ RIIB; Also referring to people such as Armour, PCT publication number WO99/58572, people such as Idusogie, WO 99/51642, and people such as Deo, U.S.6,395,272; The content of described reference is incorporated into herein with its integral body).Also known in the art modification Fc district to Fc γ RIIB in conjunction with the method for affinity (for example: people such as Ravetch reduces, U.S. Patent Publication No. 20010036459 and PCT publication number WO 01/79299, the content of described reference is incorporated into herein with its integral body).Also described district, comprised and (for example: people such as Stavenhagen, PCT publication number WO 2004/063351 have enhanced for the modified antibodies in the variant Fc district of the binding affinity of Fc γ RIIIA and/or Fc γ RIIA with respect to wild-type Fc; The content of described reference is incorporated into herein with its integral body).

Found at least four kinds of dissimilar Fc γ R, it is known as Fc γ RI (CD64), Fc γ RII (CD32), Fc γ RIII (CD16) and Fc γ RIV respectively.In human body, Fc γ RII and Fc γ RIII further are divided into Fc γ RIIa and Fc γ RIIb respectively, and Fc γ RIIIa and Fc γ RIIIb.Fc γ R is the membranin of contactin, Fc γ RII, Fc γ RIII and Fc γ RIV have a α chain, it has the extracellular region that comprises two immunoglobulin (Ig) analog structure territories, Fc γ RI has a α chain as moiety, it has the extracellular region that comprises three immunoglobulin (Ig) analog structure territories, and this α chain is relevant in conjunction with activity with IgG.In addition, Fc γ RI and Fc γ RIII have γ chain or the ζ chain as moiety, it has the signal transduction functionality (Annu.Rev.Immunol relevant with the α chain, 18,709 (2000), Annu.Rev.Immunol, 19,275 (2001)) people such as .Bruhns, Clin.Invest.Med. (Canada) 27:3D (2004) has described Fc γ RIV.

For the ADCC activity of the anti-CD 19 antibodies of measuring research, available external ADCC test, for example U.S. Patent number 5,500, and 362 or 5,821, described in 337.The useful effector cell of this test is comprised peripheral blood monocyte (PBMC) and natural killer (NK) cell.For example: can measure any special antibody and mediate target cell dissolved ability by complement activation and/or ADCC.The cell that makes research is at growth in vitro and mark; To with immunocyte bonded cell culture medium in add antibody, it can be activated by immune complex; That is: the effector cell participates in the ADCC reaction.Also can measure the complement activity of antibody.In both cases, all detect the cytolysis of target cell by from dissolved cell, removing mark.In fact, antibody can be screened in patient's oneself the serum source as complement and/or immunocyte.Can in to particular patient, use to therapeutic the antibody of energy Mediated Human ADCC in vitro tests then.Optionally, or replenish ground, can measure the ADCC activity of the molecule of research in vivo, for example: in animal model, for example people such as Clynes, the disclosed content of PNAS (USA) 95:652-656 (1998).In addition, be known in the art the ADCC level of adjustment (promptly increase or reduce) antibody, the active and active method of apoptosis optionally of CDC optionally.Referring to, for example: U.S. Patent number 6,194,551 (referring to, for example: people such as Chaouchi, J.Immunol.154 (7): 3096-104 (1995); People such as Pedersen, Blood, 99 (4): 1314-1318 (2002); People such as Alberts, MolecularBiology of the Cell; People such as Steensma, Methods Mol Med.85:323-32, (2003)).Antibody of the present invention preferably can or modified and have an ability that causes ADCC and/or CDC and/or apoptosis reaction.Preferably, can be by the test of this mensuration ADCC function, the personnel selection effector cell measures people's ADCC function.

5.3.3 immune conjugate and fusion rotein

According to some aspect of the present invention, therapeutical agent or toxin can be connected to use in the compositions and methods of the invention chimeric, the people's or humanized anti-CD 19 antibodies.In certain embodiments, can produce these conjugates (referring to the 5.1.8 joint) as fusion rotein.The example of therapeutical agent and toxin include but not limited to, the member of enediyne quasi-molecule family, for example: calicheamycin (calicheamicin) and Ai Sibo mycin (esperamicin).Also can from duocarmycin, methotrexate, Zorubicin, Melphalan, Chlorambucil, cytosine arabinoside, desacetyl vinblastine amide, zilimeisu, cis platinum, etoposide, bleomycin and 5 FU 5 fluorouracil form group obtain chemical toxicant (referring to, for example: U.S. Patent number 5,703,080 and U.S. Patent number 4,923,990).The example of chemotherapeutics also comprises Zorubicin, doxorubicin, 5 FU 5 fluorouracil, cytosine arabinoside (cytosine arabinoside), endoxan, thiotepa, taxotere (Japanese yew terpene), busulfan, endoxan, taxol, methotrexate, Platinol, Melphalan, vincaleucoblastine, bleomycin, etoposide, ifosfamide, zilimeisu, mitoxantrone, vincristine(VCR), Vinorelbine, carboplatin, Vumon, Rubomycin C, carminomycin, aminopterin, gengshengmeisu, mitomycin, Ai Sibo mycin (esperamicin) (referring to, U.S. Patent number 4,675,187), Melphalan and other relevant nitrogen mustardses.

For example, in other embodiments, can in combination therapy of the present invention, use " CVB " (1.5g/m ²Endoxan, 200-400mg/m ²Etoposide and 1 50-200mg/m ²Carmustine).CVB is the dosage regimen (people such as Patti, Eur.J.Haematol.51:18 (1 993)) that is used for the treatment of the non-Hodgkin lymphomas.Be known in the art the dosage regimen of other suitable combined chemotherapy.Referring to, for example: people such as Freedman, " Non-Hodgkin ' s Lymphomas " among the Cancer Medicine, Volume 2,3rd Edition, people such as Holland (eds.), pp.2028-2068 (Lea﹠amp; Febiger 1993).As an illustration, the first-generation chemotherapy dosage regimen of treatment moderate non-Hodgkin lymphomas comprises C-MOPP (endoxan, vincristine(VCR), procarbazine and prednisone) and CHOP (endoxan, Zorubicin, vincristine(VCR) and prednisone).Useful s-generation chemotherapy dosage regimen is m-BACOD (methotrexate, bleomycin, AC, vincristine(VCR), dexamethasone and a formyl tetrahydrofolic acid), and suitable third generation dosage regimen is MACOP-B (methotrexate, AC, vincristine(VCR), prednisone, bleomycin and a formyl tetrahydrofolic acid).Other effective medicine comprises phenyl butyrate and brostatin-1.

Other toxin of available comprises poisonous Sugar receptors, plant poison for example ricin, toxalbumin, modeccin, Toxins, botulin and diphtheria toxin in immune conjugate of the present invention.Certainly, also the complex body of various toxin can be connected in an antibody molecule, thereby regulate variable cytotoxicity.The example that is fit to be applied to the toxin of conjoint therapy of the present invention is ricin, toxalbumin, rnase, DNaseI, staphyloentero-toxin-A, pokeweed antiviral protein, spends more white tree inhibitor (gelonin), diphtheria toxin, Rhodopseudomonas extracellular toxin and pseudomonas intracellular toxin.Referring to, for example: people such as Pastan, Cell, people such as 47:641 (1986) and Goldenberg, Cancer Journal for Clinicians, 44:43 (1994).Spendable enzymatic activity toxin and fragment thereof comprise diphtheria A chain, the non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-Zhou Qujunsu, tung oil tree albumen, alizarin albumen, dyers' grapes albumen (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, spend more white tree inhibitor (gelonin), mitogellin, restrictocin, phenomycin, enomycin and tricothecene.Referring to, for example: on October 28th, 1993 disclosed WO 93/21232.

At Remington ' s Pharmaceutical Sciences, 19th Ed. (Mack PublishingCo.1995) and Goodman And Gilman ' s The Pharmacological Basis ofTherapeutics have described suitable toxin and chemotherapeutics among the 7th Ed. (MacMillan Publishing Co.1985).Those skilled in the art known other suitable toxin and/or chemotherapeutics.

Also can anti-CD 19 antibodies of the present invention be used for ADEPT by antibody being connected to the prodrug activation enzyme that prodrug (for example: peptidyl chemotherapeutics, referring to WO81/01145) is converted to the active anticancer medicine.Referring to, for example: WO 88/07378 and U.S. Patent number 4,975,278.The enzyme component of the useful immune conjugate of ADEPT comprised anyly can act on prodrug, and be translated into the enzyme that it more activated, had more cytotoxic form by this approach.

Useful in the method for the invention enzyme include but not limited to, and converts the useful alkaline phosphatase of free drug to comprising phosphatic prodrug; The prodrug that will comprise vitriol is converted to the useful aryl sulphatase of free drug; To nontoxic 5-flurocytosine being converted to the useful Isocytosine deaminase of cancer therapy drug 5 FU 5 fluorouracil; Protease, for example Serratia Proteases, thermolysin, subtilisin, carboxypeptidase and kethepsin (for example: cathepsin B and L), to convert free drug to useful to comprising the propeptide medicine for it; D-alanyl carboxypeptidase, it is useful to the prodrug that conversion comprises D-aminoacid replacement base; To glycosylated prodrug being converted to the useful lytic enzyme of free drug, for example beta-galactosidase enzymes and neuraminidase; Medicine with α-lactam derivative is converted to the useful β-Nei Xiananmei of free drug; Penicillin amidase, for example penicillin v Ntn hydrolase or penicillin G Ntn hydrolase, it is useful respectively the amine nitrogen institute deutero-medicine with phenoxy group ethanoyl or phenylacetyl to be converted to free drug.Optionally, the antibody of enzymic activity is arranged, is also referred to as " abzyme " in the art, can be used for prodrug of the present invention convert to the free active medicine (referring to, for example: Massey, Nature 328:457-458 (1987)).Can be according to the abzyme conjugate for preparing described herein, according to hope abzyme is transferred to by the human body of Malignant B cell infection.

Available technology known in the art for example uses isodigeranyl function cross-linking reagent mentioned above that enzyme of the present invention is covalently bonded in antibody.Optionally, use recombinant DNA technology known in the art, structure comprises the fusion rotein of the antigen binding domain of at least one antibody of the present invention, described antigen binding domain link to each other with at least one functional activated partial of enzyme of the present invention (referring to, for example: people such as Neuberger, Nature, 312:604-608 (1984)).

The covalent modification thing of anti-CD 19 antibodies of the present invention within the scope of the present invention.As applicable, can prepare them by the chemosynthesis of antibody or by enzyme or chemical cracking.By the target amino acid residue of antibody and tissue derived reagent are reacted, the covalent modification of other kind of anti-CD 19 antibodies is introduced molecule, described tissue derived reagent can with the side chain of selecting or N-or the reaction of C-terminal residue.

Usually with cysteinyl residue and α-salt acetate (with corresponding amine), for example Mono Chloro Acetic Acid or chlor(o)acetamide react, and obtain the derivative of carboxymethyl or Carboxylamide ylmethyl.Similarly, also can use iodo reagent.Also can by with bromine trifluoroacetone, α-bromine dimethoxy-β-(5-imidazolyl (imidozoyl)) propionic acid, chloracetyl phosphoric acid salt, N-alkyl maleimide base, 3-nitro-2-pyridyl disulfide, methyl-2-pyridyl disulfide, p-parachloromercuribenzoate, 2-chloromercuri-4-nitrophenols or chloro-7-oil of mirbane-2-oxygen-1, the 3-diazole reacts and derives cysteinyl residue.

By reacting the histidyl-residue of deriving, because this reagent is special relatively to the histidyl-side chain at pH5.5-7.0 and diethylpyrocarbonate.Offside-bromine dimethoxy bromoacetophenone is also useful; Preferably at pH6.0, under the condition of the sodium dimethylarsonate of 0.1 M, react.

With lysyl and n terminal residue and Succinic anhydried or with other carboxylic acid anhydride reaction.The effect that has the electric charge that changes lysyl-residue with deriving of carrying out of these reagent.Other suitable reagent that comprises alpha-amino residue and/or comprise the residue of epsilon-amino that is used to derive comprises imido-ester, for example pyridine azomethine acid formicester (methyl picolinimidate), pyridoxal phosphate, pyridoxal, chlorine hydroborate, trinitro-benzene-sulfonic acid, 0-methyl-isourea, 2,4-2, the 4-diacetylmethane, and carry out the catalytic reaction of transaminase with glyoxylate.

By with one or several reagent commonly used, comprising phenylglyoxal, dimethyl diketone, 1,2-hexanaphthene and ninhydrin reaction are modified the arginyl residue.The derivatization of arginyl residue requires to react under alkaline condition usually, because the high pKa of guanidine functional group.In addition, can be with the epsilon-amino group and the arginine epsilon-amino radical reaction of these reagent and Methionin.

Can in the tyrosyl residue, introduce the spectrum mark by reacting, prepare the tyrosyl residue that specificity is modified with aromatic diazonium compound or tetranitromethane.Usually, form O-ethanoyl tyrosyl class and 3-nitro-derivative respectively with N-acetyl imidazole and tetranitromethane.With ¹²⁵I or ¹³¹I iodate tyrosyl residue prepares the labelled protein that is used for the radioimmunoassay test.

By reacting with carbodiimide (R-N=C=N-R '); modify carboxyl side group (aspartyl or glutamyl) selectively; wherein R is different alkyl with R '; for example: 1-cyclohexyl-3-(4-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3-(4-nitrogen-4,4-dimethylmethane) carbodiimide.In addition, by reacting aspartyl and paddy acyl group are converted into asparagus fern acyl and glutaminyl residue with ammonium.

Often respectively glutaminyl and asparagus fern acyl residue are deaminized, form corresponding paddy acyl group and aspartoyl residue.Under neutrality or primary condition, these residues are deaminized.The form of deaminizing of these residues falls into scope of the present invention.

Other modification comprises the hydroxylation of proline(Pro) and Methionin, the phosphorylation of the oh group of seryl or threonyl residue, alpha-amino (the T.E.Creighton that methylates of Methionin, arginine and Histidine side chain, Proteins:Structure and Molecular Properties, W.H.Freeman﹠amp; Co.San Francisco, pp.79-86 (1983)), the acetylize of N-terminal amino group and the amidation of any C-terminal carboxyl groups.

The another kind of type that covalency is revised comprises chemically or enzymatic ground is attached to glucoside on the antibody.The advantage of these steps is that it does not require in the glycosylation that N-or O-are connected has the host cell of glycosylation ability and produces antibody.Rely on used combination, sugar can be attached to (a) arginine and Histidine, (b) free carboxyl group, (c) free sulfhydryl base, for example: the free sulfhydryl base of halfcystine, (d) free hydroxyl group, for example: the free hydroxyl group of Serine, Threonine or oxyproline, (e) aromatic residue, for example: the aromatic residue of phenylalanine, tyrosine or tryptophane or (f) amide group of glutamine.At disclosed WO 87/05330 on September 11st, 1987, and at Aplin and Wriston, CRC Crit.Rev.Biochem.pp.259-306 has described these methods in (1981).

5.4 the prescription of medicine, administration and dosage

Formula of medicine of the present invention comprises as the people of activeconstituents, humanized or chimeric anti-CD 19 antibodies.Prescription comprises and is suitable for the weight or meausurement unit that human patients is given and produces the naked antibody of the significant quantity of the reaction of wanting, immune conjugate or fused protein, preferred disinfectant.For example, can come assaying reaction, for example, but be not limited only to, circulation B cell consumption, organize disappearing or the minimizing of disease symptoms of B cell consumption, B cell malignancies by the physiologic effect of measuring antagonism CD19 antibody component.Those of ordinary skills know other test, and it can be used for the level of assaying reaction.

5.4.1 the prescription of medicine

The composition of available pharmaceutically acceptable carrier preparation anti-CD 19 antibodies.Term " pharmaceutically acceptable " refers to not influence one or more non-toxic materials of the biological activity effect of activeconstituents.This preparation can routine comprises salt, buffer reagent, sanitas, compatible vector and other therapeutical agent optionally.This pharmaceutically acceptable preparation also can conventional comprise compatible solid or liquid filler, thinner or be suitable for capsule material to people's administration.When being used for medical science, salt should be pharmaceutically acceptable, but also non-pharmacy acceptable salt can be advantageously used in the preparation of its pharmacy acceptable salt, and should not be excluded outside scope of the present invention.This pharmacology and pharmacy acceptable salt include but not limited to, by the prepared salt of following acid: hydrochloric acid, Hydrogen bromide, sulfuric acid, nitric acid, phosphoric acid, toxilic acid, acetic acid, Whitfield's ointment, citric acid, boric acid, formic acid, propanedioic acid, succsinic acid etc.Simultaneously, pharmacy acceptable salt can be made basic metal or alkaline earth salt, for example salt of sodium, potassium or calcium.Lived or the abiotic composition of term " carrier " expression, nature or synthetic, combine with activeconstituents with it and to promote application.The composition of composite medicine can not weaken the mode intermingling that expected drug is renderd a service with the blended interaction basically with antibody of the present invention yet.

According to some aspect of the present invention, antibody that can be by will having requirement purity or immune conjugate mix the composition (Remington ' sPharmaceutical Sciences of the anti-CD 19 antibodies that the form with freeze-dried formulation or aqueous solution of preparing is used to store with optional physiologically acceptable carrier, vehicle or stablizer, 16th edition, Osol, A.Ed. (1999)).Acceptable carrier, vehicle or stablizer are nontoxic to the recipient under used dosage and concentration, and it comprises buffer reagent, for example phosphoric acid salt, Citrate trianion and other organic acids; Antioxidant, it comprises xitix and methionine(Met); Sanitas (for example: stearyl dimethyl benzyl ammonium chloride; Hexamethonium chloride; Benzalkonium chloride, Solamin; Phenylic acid, butyl or phenylcarbinol; Alkyl p-hydroxybenzoic acid, for example methyl or propylparaben; Catechol; Resorcinol; Hexalin; 3-amylalcohol and meta-cresol); The polypeptide of lower molecular weight (less than about 10 residues); Protein, for example: serum albumin, gelatinum or immunoglobulin (Ig); Hydrophilic polymer, for example: polyvinylpyrrolidine; Amino acid, for example: glycine, glutamine, asparagine, Histidine, arginine or Methionin; Monose, disaccharides and other carbohydrate comprise glucose, seminose or dextrin; Sequestrant, for example: EDTA; Sugar, for example: sucrose, N.F,USP MANNITOL, trehalose or Sorbitol Powder; Anti-salt forms ion, for example: sodium; Metal composite (for example zinc-albumen composition) and/or nonionic surface active agent, for example: TWEEN, PLURONICS ^TMOr polyoxyethylene glycol (PEG).

The composition of anti-CD 19 antibodies also optionally comprises suitable preservatives, for example: benzalkonium chloride; Butylene-chlorohydrin; P-hydroxybenzoic acid and Thiomersalate.

The composition of anti-CD 19 antibodies can be present in the unit dosage routinely, and can prepare by known any method in the pharmaceutical field.All methods all comprise promoting agent and the carrier of being made up of one or more submembers blended step mutually.Normally, prepare composition, then, in case of necessity, make the product shaping by evenly and closely active substance and liquid carrier, ground solid-state carrier or both being mixed mutually.

The composition that is suitable for administered parenterally comprises the moisture or anhydrous preparation of disinfectant of anti-CD 19 antibodies routinely, its preferably with the blood samples of patients equipressure.Can utilize suitable dispersion or wetting agent and suspension agent to prepare said preparation in accordance with known methods.The sterilization injection is disinfectant Injectable solution or nontoxic parenteral acceptable diluent or the suspension of solvent also, for example: 1, the solution of 3-butanediol.Used acceptable carrier and solvent are water, RingerShi solution and isoosmotic sodium chloride solution.In addition, the disinfectant fixed oil is used as traditionally the medium of solvent or suspension.For this purpose, can use the fixed oil of any gentleness, comprise synthetic list-or two-glyceryl ester.In addition, available lipid acid, for example oleic acid preparation injection formulations.The carrier formulation that is suitable for injections such as oral area, subcutaneous, intravenously, intramuscular can be at Remington ' s Pharmaceutical Sciences, and Mack Publishing Co.Easton finds among the PA.In certain embodiments, be suitable for various route of administration the carrier formulation can with RITUXAN ^TMThat describes is identical or similar.Referring to: Physicians ' Desk Reference (Medical Economics Company, Inc.Montvale, NJ, 2005), pp.958-960 and 1354-1357, described reference is incorporated into herein as a reference with its integral body.In certain embodiments of the invention, prepare the composition that is used for intravenous anti-CD 19 antibodies with sodium-chlor, Trisodium citrate dihydrate, polysorbate80 and sterilized water, wherein the pH regulator of composition is about 6.5.One skilled in the art will appreciate that intravenous injection provides a kind of useful mode of injection owing to transmit the overall circulation of antibody fast.Yet intravenous injection is subjected to the restriction of vascular hamper, and described vascular hamper comprises the endotheliocyte and the interior subcutaneous matrix of vascular system.The vascular hamper is still the more significant problem of solid tumor to the therapeutic anti bulk absorption.Lymphoma has than higher blood flow rate, helps effective antibody to carry.The lymphatic vessel route of administration, for example subcutaneous or intramuscular injection, or, also provide the process useful of treatment B cell lymphoma by the lymphatic vessel catheterization.In preferred embodiments, the anti-CD 19 antibodies of the compositions and methods of the invention is subcutaneous from the body administration.In this embodiment preferred, (for example: PBS and/or Citrate trianion) comes compositions formulated with about 50mg/ml as freeze-dried reagent or in liquid buffer.

Pharmaceutical formulation herein also can comprise and surpass a kind of activeconstituents for treatment specific adaptations disease needs, preferably has not the material of the complementary activity of reverse influence mutually.For example: can use immunosuppressant preparation further is provided.This molecule suitably with to the effective dosage of intended target combines appearance.

Also activeconstituents can be put into the microcapsule of preparation, for example: by condensation technique or interfacial polymerization, for example: Walocel MT 20.000PV or gelatinum microcapsule and poly--(methyl vinylformic acid) microcapsule, respectively at colloidal drug delivery system (for example: liposome, albumin microsphere spheroid, microemulsion, receive the particle and the wafer of receiving) or in big latex.At Remington ' sPharmaceutical Sciences 16th edition, Osol, A.Ed. discloses such technology in (1980).

The preparation that is used for vivo medicine-feeding generally is a disinfectant.Can be by finishing easily with the sterilising filtration membrane filtration.

Can prepare extended release preparation.The suitable example of extended release preparation comprises the polymeric semipermeability matrix of the solid hydrophobic that comprises anti-CD 19 antibodies, and this matrix is the form with the typing thing, for example: film or micro-capsule.The example that continues release matrix comprises polyester, hydrogel (for example: many (2-hydroxyethyl-methylacrylic acids) or many (vinyl alcohols)), polylactide (U.S. Patent number 3,773,919), the L-L-glutamic acid and the multipolymer of γ ethyl-L-L-glutamic acid, nondegradable ethylene-vinyl acetate, degradable poly lactic coglycolic acid, for example LUPRON DEPOT ^TM(the injectable microsphere of forming by poly lactic coglycolic acid and leuprolide acetate) and poly--D-(-)-3-hydroxybutyric acid.When polymer when for example ethylene-vinyl acetate and lactic-co-glycolic acid can discharge molecule and surpass 100 days, some hydrogel can discharge albumen in a short time.When encapsulated antibody kept for a long time in vivo, they may be exposed to humidity and cause sex change or polymerization under 37 ℃, cause bioactive forfeiture and immunogenic may the change.Mechanism according to relating to can obtain to realize stable reasonable strategy.For example: if find aggregation of multiple is to exchange the intermolecular S-S key that forms by thioaldehydes, then can by revise sulfhydryl residue, from the acidic solution freeze-drying, control water capacity, utilize suitable additive, and develop special polymeric matrices composition and realize stablizing.In some concrete embodiment, the used pharmaceutically acceptable carrier of composition of the present invention does not influence people's ADCC or CDC.

The composition of anti-CD 19 antibodies disclosed herein also can be made immune liposome and prepare." liposome " is by being used for carrying medicine (for example: the folliculus that various types of liposomes, phosphatide and/or tensio-active agent anti-CD 19 antibodies disclosed herein) formed to the mankind.The component of liposome is the bilayer form usually, is similar to the spread pattern of liposome on microbial film.Available method known in the art preparation comprises the liposome of antibody of the present invention, for example: people such as Epstein, Proc.Natl.Acad.ScL USA, 82:3688 (1985); People such as Hwang, Proc.Natl.Acad.Sci.USA, 77:4030 (1980); And U.S. Patent number 4,485,045 and 4,544,545 is described.At U.S. Patent number 5,013, the liposome with longer cycling time is disclosed in 556.Can pass through reverse phase evaporation, prepare useful especially liposome with the lipid composition that comprises phosphatidylcholine, cholesterol and PEG-deutero-phosphatidylethanolamine (PEG-PE).Strainer by the predetermined hole diameter size filters liposome, produces the liposome of wanting diameter.Antibody of the present invention can combine with liposome via the disulphide mutual exchange reaction, and as people such as Martin, J.Biol.Chem.257:286-288 (1982) is described.Also can comprise therapeutical agent in the liposome.Referring to: people such as Gabizon, J.NationalCancer Inst. (19) 1484 (1989).

Some preferred drug substances include but not limited to:

(a) be filled in the disinfectant that is used for intravenously (i.v.) injection anti-CD 19 antibodies in the bottle of single use of 100mg (10mL) or 500mg (50mL), the concentrated solution of preservative-free with the concentration of 10mg/ml.Available sodium-chlor, Trisodium citrate dihydrate, polysorbate and the sterilized water that is used to inject are prepared product.For example: prepare product at 9.0mg/ml sodium-chlor, 7.35mg/ml Trisodium citrate dihydrate, 0.7mg/ml polysorbate80 and the sterilized water that is used for injecting.With pH regulator is 6.5.

(b) disinfectant, the lyophilized powder that are used for subcutaneous (s.c.) injection in the phial that uses separately.Available sucrose, L-histidine monohydrochloride monohydrate, L-Histidine and polysorbate 20 are prepared product.For example: the bottle of each single use can comprise 150mg anti-CD 19 antibodies, 123.2mg sucrose, 6.8mgL-histidine monohydrochloride monohydrate, 4.3mgL-Histidine and 3mg polysorbate 20.With the bottle of the single use of 1.3ml sterile water for injection reconstruct, produce about 1.5ml solution, every 1.25ml carries the antibody (100mg/ml) of 125mg.

(c) be used for the disinfectant of vein (i.v.) injection, the lyophilized powder of preservative-free.Available α-trehalose dihydrate compound, L-Histidine HCl, Histidine and polysorbate 20USP prepare product.For example: each bottle can be adorned 440mg anti-CD 19 antibodies, 400mg α, α-trehalose dihydrate compound, 9.9mgL-Histidine HCl, 6.4mgL-Histidine and 1.8mg polysorbate 20, USP.With 20ml fungistat water for injection (BWFI), comprise 1.1% phenylcarbinol and be reconstructed as the USP of sanitas, obtain the solution that pH is approximately 6 the multiple doses that comprises 21mg/ml antibody.

(d) the disinfectant lyophilized powder of intravenous infusion, anti-CD 19 antibodies is wherein prepared with sucrose, polysorbate, monobasic sodium phosphate monohydrate and Di-Sodium Phosphate dihydrate.For example: each bottle that uses separately can be adorned 100mg antibody, 500mg sucrose, 0.5mg polysorbate80,2.2mg monobasic sodium phosphate monohydrate and 6.1mg Di-Sodium Phosphate dihydrate.There is not sanitas.Then with 10ml sterile water for injection, USP reconstruct, the pH that obtains is about 7.2.

(e) solution that is used for hypodermic disinfectant, preservative-free in the 1ml prefilled syringe that uses separately.Available sodium-chlor, monobasic sodium phosphate dihydrate, Di-Sodium Phosphate dihydrate, Trisodium Citrate, citric acid monohydrate compound, N.F,USP MANNITOL, polysorbate80 and water for injection, USP prepare product.Can add sodium hydroxide pH is adjusted into about 5.2.

For example: the pharmaceutical product that can prepare each syringe output 0.8ml (40mg).Every 0.8ml comprises 40mg anti-CD 19 antibodies, 4.93mg sodium-chlor, 0.69mg monobasic sodium phosphate dihydrate, 1.22mg Di-Sodium Phosphate dihydrate, 0.24mg Trisodium Citrate, 1.04 citric acid monohydrate compounds, 9.6mg N.F,USP MANNITOL, 0.8mg polysorbate80 and water for injection, USP.

(f) in the bottle that uses separately, be used for subcutaneous (s.c.) injection, and with the disinfectant of sterile water for injection (SWFI), USP reconstruct, the lyophilized powder of preservative-free.Available sucrose, histidine monohydrochloride monohydrate, L-Histidine and tween preparation product.For example: the 75mg bottle can be adorned anti-CD 19 antibodies, 93.1mg sucrose, 1.8mgL-histidine monohydrochloride monohydrate, 1.2mgL-Histidine and the 0.3mg polysorbate 20 of 129.6mg or 112.5mg, and with its be designed to 0.9mlSWFI, USP reconstruct after, the antibody of 75mg among the output 0.6ml.The 150mg bottle can be adorned 202.5mg or 175mg anti-CD 19 antibodies, 145.5mg sucrose, 2.8mgL-histidine monohydrochloride monohydrate, 1.8mgL-Histidine and 0.5mg polysorbate 20, and with its be designed to 1.4mlSWFI, USP reorganization after, the antibody of 150mg among the output 1.2ml.

(g) with disinfectant sterilized water reconstruct, that be used to inject, lyophilized products.Available N.F,USP MANNITOL, Histidine and glycine are prepared the product that is used for muscle (IM) injection, with the small bottle packing that uses separately.For example: each bottle that uses separately can be adorned 100mg antibody, 67.5mg N.F,USP MANNITOL, 8.7mg Histidine and 0.3mg glycine, when being designed to it with the reconstruct of 1.0ml Injectable sterile water, can export the antibody of 100mg among the 1.0ml.Optionally, each makes the land used bottle can adorn 50mg antibody, 40.5mg N.F,USP MANNITOL, 5.2mg Histidine and 0.2mg glycine separately, when being designed to it with the reconstruct of 0.6ml Injectable sterile water, can export the antibody of 50mg.

(h) be used for the disinfectant of muscle (IM) injection, the solution of preservative-free, its administration concentration is 100mg/ml.Can in the bottle that uses separately, prepare product with Histidine, glycine and Injectable sterile water.For example: each bottle that uses separately can be mixed with the 1.2ml volume with 100mg antibody, 4.7mg Histidine and 0.1mg glycine, is designed to transmit among the 1ml 100mg antibody.Optionally, each bottle that uses separately can be mixed with 0.7ml or 0.5ml volume with 50mg antibody, 2.7mg Histidine and 0.08mg glycine, is designed to transmit among the 0.5ml 50mg antibody.

In certain embodiments, pharmaceutical composition of the present invention is stable down at 4 ℃.In certain embodiments, pharmaceutical composition of the present invention is at room temperature stable.

5.4.2 the half life of antibody

In certain embodiments, the half life of the anti-CD 19 antibodies of the compositions and methods of the invention, be at least about 4 to 7 days.In certain embodiments, the average half life of the anti-CD 19 antibodies of the compositions and methods of the invention, be at least about 2 to 5 days, 3 to 6 days, 4 to 7 days, 5 to 8 days, 6 to 9 days, 7 to 10 days, 8 to 11 days, 8 to 12 days, 9 to 13 days, 10 to 14 days, 11 to 15 days, 12 to 16 days, 13 to 17 days, 14 to 18 days, 15 to 19 days or 16 to 20 days.In other embodiment, the half life of the anti-CD 19 antibodies of the compositions and methods of the invention, can reach about 50 days.In certain embodiments, available methods known in the art prolong the half life of the antibody of the compositions and methods of the invention.This prolongation can reduce the total amount and/or the frequency of the dosage of antibody compositions of the present invention successively.In U.S. Patent number 6,277,375; Antibody that has prolonged half life in the body and preparation method thereof is disclosed among international publication number WO 98/23289 and the WO 97/3461.

Also can by with the inert polymer molecule attached to having or not having on the antibody of multi-functional connexon, be connected with the N-of antibody or the locus specificity of C-end by PEG, or by the epsilon-amino group on lysyl-residue, prolong anti-CD 19 antibodies of the present invention serum circulation in vivo, described inert polymer molecule such as high molecular weight polyethylene glycol (PEG).Employing causes the line style or the effect of ramose polymer-derived of biological activity minimum loss.Available SDS-PAGE and mass spectrometry are paid close attention to combination degree, guarantee that the PEG molecule is connected with the suitable of antibody.Available size-eliminating or unreacted PEG is removed from antibody-PEG conjugate with ion exchange chromatography.Available method known to those skilled in the art, immunoassay for example described herein, detect PEG derive antibody in conjunction with rendeing a service in activity and the body.

In addition, can be with the antibody and the albumin bound of the compositions and methods of the invention, more stable in vivo or have in vivo longer half life in order to make antibody.Be known in the art these methods, referring to, for example: international publication number WO 93/15199, WO 93/15200 and WO01/77137; European patent number EP 413,622, described reference is incorporated into herein as a reference.

5.4.3 administration and dosage

Can be by any approach with composition of the present invention to the human patients administration, include but are not limited to: intradermal, skin, subcutaneous, muscle, suction (for example via aerosol), cheek or mouthful (for example hypogloeeis), part (are skin and mucomembranous surface, comprise the tracheae surface), in the sheath, in the intraarticular, pleura, in the brain, in the intra-arterial, abdomen, in the oral area, lymph, in term, the administration of rectum or vagina, by local catheter infusion, or by direct intralesional injection.In preferred embodiments, carry out administration with intravenously propelling or intravenous fluids (for example: 0.5 to 2 hour) in specific time with composition of the present invention.Can carry composition of the present invention by the mode of wriggling or the form of storage, but anyly depend on to optimal approach under the stable condition, known in the art, the character (that is: dosage, prescription) of the character of the general status of species, age, sex and object, treatment situation and the particular composition of severity and/or institute's administration.In specific embodiment, route of administration is for by the continuous transfusion in bolus or certain period, a Monday or twice.In one embodiment, be that administration is carried out with composition of the present invention and/or method in the basis with out-patient.

In certain embodiments, the dosage that comprises the composition of anti-CD 19 antibodies is that unit calculates with weight in patients mg/kg.In other embodiment, the dosage that comprises the composition of anti-CD 19 antibodies is that unit calculates with patient's fat-free mass (that is: body weight deducts body fat content) mg/kg.In other embodiments, comprise the dosage of composition of anti-CD 19 antibodies with patient's body surface area mg/m ²For unit calculates.In other embodiments, the dosage that comprises the composition of anti-CD 19 antibodies is that unit calculates with every dose of milligram number to patient's administration.The mensuration of dosage can be used with the compositions and methods of the invention, and dose unit can be changed with the standard manner in this area.

Those skilled artisans will appreciate that can be according to the situation that comprises age, sex, species and object (for example: the stage of B cell malignancies), the desirability of cell consumption, the disease that will treat and/or many factors employed and confirmable specific antibodies of those skilled in the art or Fab select dosage.For example: the dose response curve that can derive from vitro test system or animal model (for example: cotton mouse or monkey) test macro is known the significant quantity of the present composition by inference.Be known in the art the model of assessment antibody influence and method (people such as Wooldridge, Blood, 89 (8): 2994-2998 (1997), incorporate into herein as a reference) with its integral body.In certain embodiments, for the particular B cell malignancies, the treatment plan that is used for antibody therapy of standard uses the compositions and methods of the invention in available this area.

The example of the dosage of the treatment plan that uses in the method for the present invention include but not limited to, once a day, on every Wendesdays time (intermittent), weekly or per 14 days once.In certain embodiments, the dosage of treatment plan include but not limited to, every month the dosage or the dosage in every 6-8 week.

Those skilled in the art can understand, and compare with keeping treatment plan, and higher dosage and/or higher administration frequency are used in the initial stage treatment usually.

In embodiments of the invention, anti-CD 19 antibodies is attached on the B cell, can causes more effective (that is: low dosage) B cell consumption (as described here) like this.The combination of higher degree can be realized in the high ground of people CD19 density on the patient B cell surface.In an embodiment, the dosage of antibody (choosing wantonly in the pharmaceutically acceptable carrier as a composite medicine part) is minimum is about 0.0005,0.001,0.05,0.075,0.1,0.25,0.375,0.5,1,2.5,5,10,20,37.5 or 50mg/m ²And/or less than about 500,475,450,425,400,375,350,325,300,275,250,225,200,175,150,125,100,75,60,50,37.5,20,15,10,5,2.5,1,0.5,0.375,0.1,0.075 or 0.01 mg/m ²In certain embodiments, dosage is about 0.0005 to about 200mg/m ²Between, about 0.001 to 150mg/m ²Between, about 0.075 to 125mg/m ²Between, about 0.375 to 100mg/m ²Between, about 2.5 to 75mg/m ²Between, about 10 to 75mg/m ²Between, about 20 to 50mg/m ²Between.In relevant embodiment, the dosage of used anti-CD 19 antibodies is at least about 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1,1.5,2,2.5,3,3.5,4,4.5,5,5.5,6,6.5,7,7.5,8,8.5,9,9.5,10,10.5,11,11.5,12,12.5,13,13.5,14,14.5,15,15.5,16,16.5,17,17.5,18,18.5,19,19.5,20, the 20.5mg of every kg weight in patients.In certain embodiments, the dosage of used naked anti-CD 19 antibodies is at least every kg weight in patients about 1 to 10,5 to 15,10 to 20 or 15 to 25mg.In certain embodiments, the dosage of used anti-CD 19 antibodies is at least every kg weight in patients about 1 to 20,3 to 15 or 5 to 10mg.In preferred embodiments, the dosage of used anti-CD 19 antibodies is at least every kg weight in patients about 5,6,7,8,9 or 10mg.In certain embodiments, the single dose unit of antibody (randomly in the pharmaceutically acceptable carrier as a pharmaceutical composition part) can be about at least 0.5,1,2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,200,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248, or 250 micrograms/m ²In other embodiment, dosage can reach every 1g of single dose unit.

Above-mentioned dosage all is exemplary, and can be used for and the combining of the compositions and methods of the invention, yet when uniting with anti-CD 19 antibodies and toxin or radiation treatment reagent when using, preferred above-mentioned than low dosage.In certain embodiments, when the patient has low-level CD19 density, preferred above-mentioned than low dosage.

In certain embodiments of the invention, when using chimeric anti-CD 19 antibodies, the dosage of chimeric antibody or total amount should be greater than every kg weight in patients about 2,3,4,5,6,7,8,9,10,11,12,13,14,15 or 16mg.In other embodiments of the present invention, when using chimeric anti-CD 19 antibodies, the dosage of chimeric antibody or total amount should be less than every kg weight in patients about 1,0.9,0.8,0.7,0.6,0.5,0.4,0.3,0.2 or 0.1mg.

In some embodiment of method of the present invention, the dosage of antibody of the present invention and/or composition is lower than about 375mg/m ²Dosage is lower than about 37.5mg/m ²Dosage should be lower than about 0.375mg/m ²And/or dosage should be lower than about 0.075mg/m ²And about 125mg/m ²In the preferred embodiment of the inventive method, dosage regimen comprises low dosage, recurrence interval administration.For example: in one embodiment, the dosage of composition of the present invention can be lower than about 375mg/m every about 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50,60,70,80,90,100,125,150,175 or 200 day ²

With the philtrum of the compositions and methods of the invention treatment, specific dosage can cause the B cell consumption, and administration time was at least about 1,2,3,5,7,10,14,20,30,45,60,75,90,120,150 or 180 day or longer.In certain embodiments, pre-B cell (not expressing surface immumoglobulin) is consumed.In certain embodiments, mature B cell (expression surface immumoglobulin) is consumed.In other embodiment, the non-pernicious type of all of B cell can show consumption.The B cell of available any of these type is measured the B cell consumption.The B cell consumption can be in body fluid such as serum, or measure in as marrow at tissue.In the preferred embodiment of the inventive method, the b cell level with respect to using the preceding patient of synthetics of the present invention and method treatment consumes at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% B cell.In the preferred embodiment of the inventive method, with respect to the typical standard b cell level of people, the emptying of B cell is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.In relevant embodiment, with considering the typical standard b cell level of patient's comparative determination people that age, sex, body weight and other factor are treated.

In certain embodiments of the invention, dosage is that about 125mg/m2 or antibody still less or Fab cause B cell consumption in about at least 7,14,21,30,45,60,90,120,150 or 200 days.In another typical embodiment, about 37.5mg/m ²Or dosage still less causes B cell consumption in about at least 14,21,30,45,60,90,120,150 or 200 days.In other embodiment, about 0.375mg/m ²Or dosage still less causes B cell consumption in about at least 7,14,21,30,45 or 60 days.In another embodiment, about 0.075mg/m ²Or dosage still less causes B cell consumption in about at least 7,14,21,30,45,60,90,120,150 or 200 days.In other embodiment, approximately 0.01mg/m2,0.005mg/m2 or even 0.001mg/m2 or dosage still less cause B cell consumption in about at least 3,5,7,10,14,21,30,45,60,90,120,150 or 200 days.According to these embodiments, can be by any suitable approach with dosed administration, but can randomly pass through the subcutaneous route administration.

On the other hand, the present invention finds to use than the employed antibody of the method for present employing or the lower dosage of antibody fragment and realizes the B cell consumption and/or to the treatment of B cell disorder.Like this, in another embodiment, the invention provides the method for a kind of B of consumption cell and/or treatment B cell disorder, comprise the specific combination of significant quantity in the antibody of CD19 to the human body administration, wherein said dosage is approximately 500,475,450,425,400,375,350,325,300,275,250,225,200,175,150,125,100,75,60,50,37.5,20,10,5,2.5,1,0.5,0.375,0.25,0.1,0.075,0.05,0.001,0.0005mg/m ²Or still less, cause about at least 3,5,7,10,14,21,30,45,60,75,90,120,150,180, or B cell consumption (the B cell of circulation and/or tissue) 25%, 35%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or more in 200 days or longer period.In typical embodiment, about 125mg/m ²Or 75mg/m ²Or dosage still less can cause in about at least 7,14,21,30,60,75,90,120,150 or 180 days about at least 50%, 75%, 85% or the consumption of 90%B cell.In other embodiment, about 50,37.5 or 10mg/m ²Dosage can cause in about at least 7,14,21,30,60,75,90,120 or 180 days about at least 50%, 75%, 85% or the consumption of 90%B cell.In other other embodiment, about 0.375 or 0.1mg/m ²Dosage can cause in about at least 7,14,21,30,60,75 or 90 days about at least 50%, 75%, 85% or the consumption of 90%B cell.In other embodiments, about 0.075,0.01,0.001 or 0.0005mg/m ²Dosage can cause in about at least 7,14,21,30 or 60 days about at least 50%, 75%, 85% or the consumption of 90%B cell.

In certain embodiments of the invention, can improve or reduce dosage, for example but be not limited only to marrow to keep the constant dosage in blood or the tissue.In relevant embodiment, improve or reduce dosage to about 2%, 5%, 8%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 95% so that the antibody of the present composition and method remains on the level that needs.

In certain embodiments, can regulate dosage and/or reduce infusion rate according to the immune response of patient the present composition and method generation.

According to an aspect of the inventive method, can at first use the input dosage of anti-CD 19 antibodies and/or composition of the present invention, use subsequently maintenance dose up to the B cell malignancies of treatment developed or course of treatment of stipulating subsequently (for example: CAMPATH ^TM, MYLOTARG ^TMOr RITUXAN ^TM, the latter can be docked the dosage that subject patient uses increases prescribed dose quantity, as the additional data that produces).

According to another aspect of the inventive method, can use the compositions and methods of the invention pretreat patient earlier, detect the minimum value of immunity reaction, or the minimal side effect of the compositions and methods of the invention.

5.4.4 toxotest

The standard medicine rules of available cell cultures or laboratory animal, tolerance, toxicity and/or effectiveness to composition of the present invention and/or treatment plan are tested, for example: measure LD50 (dosage of overall 50% mortality ratio), ED50 (overall 50% dosage of effectively treating) and IC50 (reaching 50% inhibiting effective dose).In preferred embodiments, dosage refers to reach the effective dose that at least 60%, 70%, 80%, 90%, 95% or 99% circulation B cell consumption or the consumption of circulation immunity sphaeroprotein or both consume.The dose ratio of toxic agent and therapeutical agent refers to therapeutic index, it can be expressed as the ratio of LD50/ED50.The preferred therapy that shows big therapeutic index.When use showing the therapy of toxic side effect, should note designing delivery system, this reagent is transported to the CD19 express cell, so that will drop to minimumly, and therefore reduce side effect to the potential damage of CD19 negative cells.

The dosage of employed composition of human body and/or treatment plan is formulated in available acquisition from the data of cell culture test and zooscopy.The dosage of this reagent preferably in the scope of circulation composition, comprises having a small amount of toxicity or nontoxic ED50.Dosage can change in this scope according to formulation of using and used route of administration.For any therapy of using in the inventive method, all available appropriate animal model is measured the effective dose of treatment.Kind according to animal model, dosage according to technical acceptable prescription decider body and function, for example: people such as Freireich, Quantitativecomparison of toxicity of anticancer agents in mouse, rat, monkey, dog, and human, Cancer Chemotherapy Reports, NCI 1966 40:219-244 are provided.May be useful from the data that cell culture test obtained to prediction potential toxicity.Can formulate the given dose that reaches the circulating plasma concentration range with zooscopy, described scope comprises as the IC50 that is measured in cell culture (i.e. the concentration of the inhibiting mixture of symptom that reaches half-maximum of test).Available such information is measured the dosage useful to human body more accurately.Can measure the levels of drugs of blood plasma, for example: by high performance liquid chromatography, ELISA or by test based on cell.

5.5 patient's diagnosis, stage divide and treatment plan

According to some aspect of the present invention, according to the treatment plan and the dosage of several factors selection the compositions and methods of the invention, several factors described herein includes but are not limited to, B cell disease of being treated or disorderly stage.Those skilled in the art can determine suitable treatment plan at specified phase some or a group patient B cell disease or disorder.Can make dose response curve with the standard schedule of this area, be used for measuring the significant quantity of the used composition of the present invention of the patient of treatment B cell disease or disorderly different steps.Usually, compare late period B cell disease or disorderly bigger dosage and/or the administration more continually in than long duration of needs of patients with the patient of early stage B cell disease or disorder.

Available anti-CD 19 antibodies of the present invention, composition and method treatment B cell disease comprise the B cell malignancies.Term " B cell malignancies " comprises the malignant tumour of any B of coming from cell line cell.The typical B cell malignancies comprises, but be not limited only to: B cell subsets non-Hodgkin lymphomas (NHL), it comprises rudimentary/folliculus NHL, small lymphocyte (SL) NHL, middle rank/folliculus NHL, middle rank diffusion NHL, senior immunoblast NHL, senior lymphocytoblast NHL3, senior small non-cleaved cell NHL; Mantle-cell lymphoma and huge sick NHL; Burkitt's lymphoma; Multiple myeloma; Preceding-B acute lymphoblastic leukemia and other are derived from the malignant tumour of early stage B cell precursor; Common acute Lymphocytic leukemia (ALL); Lymphocytic leukemia (CLL), it comprises the CLL and the nonmutationed CLL of immunoglobulin (Ig) of immunoglobulin (Ig) sudden change; Hairy cell leukemia; Non-acute lymphoblastic leukemia; The Fahrenheit macroglobulinemia; Diffusion large B cell lymphoid tumor (DLBCL), it comprises secondary nodules B cell sample (GCB) DLBCL, activating B cell sample (ABC) DLBCL5 and 3 type DLBCL; The prolymphocyte leukemia; Light chain disease; Plasmoma; The osteosclerosis myelomatosis; Plasma cell leukemia; The MG (MGUS) that feature is not clear; Smoulder multiple myeloma (SMM); Chronic multiple myeloma (IMM); Hodgkin's lymphomas, it comprise typical case and the trifle lymphocyte before-leading type; Lymph-plasma cell lymphoma (LPL) and marginal zone lymphoma, it comprises stomach mucous membrane bonded Lymphoid tissue (MALT) lymphoma.

The contriver has shown that antibody of the present invention and composition can consume sophisticated B cell.Therefore, on the other hand, available the present invention treats sophisticated B cell malignancies (that is: at the cell surface expression immunoglobulin (Ig)) and includes but are not limited to follicular lymphoma, mantle-cell lymphoma, burkitt's lymphoma, multiple myeloma, diffusion large B cell lymphoid tumor (DLBCL), it comprises secondary nodules B cell sample (GCB) DLBCL, activatory B cell sample (ABC) DLBCL5, and 3 type DLBCL, hodgkin's lymphomas, it comprises the preceding leading type of lymphocyte of typical and trifle, lymph-plasma cell lymphoma (LPL), marginal zone lymphoma, it comprises stomach mucous membrane association Lymphoid tissue (MALT) lymphoma, and lymphocytic leukemia (CLL), it comprises the CLL and the nonmutationed CLL of immunoglobulin (Ig) of immunoglobulin (Ig) sudden change.

In addition, CD19 is expression ratio in the development of B cell, and for example therefore CD20 early is particularly suitable for treating pre B cell and jejune B cell malignancies (that is: not expressing immunoglobulin (Ig) at cell surface), for example, and in marrow.Exemplary pre B cell and jejune B cell malignancies include but not limited to acute lymphoblastic leukemia.

In other particular, available the present invention treats the outer tumour of tubercle.

5.5.1B the diagnosis of cell malignancies and stage divide

The developmental stage of cancer, cancer described herein for example can form the B cell disease or the disorder (for example: non-Hodgkin lymphomas, diffusion large B cell lymphoid tumor, follicular lymphoma and Burkitt lymphoma) of tumour, usually the degree of propagating by health with cancer is a feature, and is divided into the stage of following four prognosis as a result usually.Phase I: cancer is positioned in the specific tissue, and is not diffused into lymphatic node.Phase: cancer is diffused into the lymphoglandula that closes on, and promptly shifts.Phase I: in lymphoglandula, find cancer away from the body region of tissue source, and opposite with a tumour, may comprise a large amount of or multiple tumour.Phase IV: cancer be diffused into health away from part.Can determine the stage of cancer by clinical observation well known to those skilled in the art and test method.The stage of above-mentioned cancer can be used for traditionally to forming the clinical diagnosis of the cancer of feature with tumour, and can be used for and interrelate with the compositions and methods of the invention treatment B cell disease and disorder.The disease that general early stage disease refers to remain positioned in the part of patient body or also do not have to shift.

For the B cell disease and the disorder of non-formation tumour, for example but be not limited only to multiple myeloma, determine the standard difference of disease stage.The widespread use Durie-Salmon stage is divided system.In this stage division system, the clinical stage of disease (Phase I, II or III) depends on several take off data, comprises quantity, oxyphorase value and the serum calcium level of the proteic level of M, dissolving bone pathology.Come the further division stage according to kidney (kidney) function (being divided into A or B).Divide system according to the Durie-Salmon stage, Phase I (low cell mass) has following whole characteristics: oxyphorase value＞10g/dL; The serum calcium value normal or≤12mg/dL; Bone x ray, normal skeletal structure (grade 0) or only single pulp glucagonoma; And low M-component productive rate: IgG value＜5g/dL, IgA value＜3g/d, Bence Jones protein＜4g/24 hour.The patient of the Phase I general not damage or the symptom of relevant organ or tissue.The characteristics of Phase (intermediate cell group) are both not meet Phase I, also do not meet Phase I.The characteristics of Phase I (high cell mass) are one or more in following: oxyphorase value＜8.5g/dL; Serum calcium value＞12mg/dL; High-grade dissolving bone pathology (scale3); High M-component productive rate: IgG value＞7g/dL, IgA value＞5g/dL, Bence Jones protein＞12g/24 hour.Subclassification (A or B), wherein A refers to that (serum creatinine value＜2.0mg/dL) and B refer to abnormal renal function (serum creatinine value＞2.0mg/dL) to relatively normal renal function.

The stage division system of another myelomatosis divides system (ISS) in the international stage of myelomatosis.This system more effectively the resolution stage divide the group, and be foundation with the 2-microglobulin (β 2-M) and the albuminous serum level of easy mensuration.ISS according to myelomatosis, the feature of Phase I is β 2-M＜3.5 and albumin 〉=3.5, and the feature of Phase is β 2-M＜3.5 and albumin＜3.5 or B2-M3.5-5.5, and the feature of Phase I is a B2-M5.5 (multiple myeloma WARF, New Canaan, CT).

The stage of patient B cell malignancies is determined by clinical.As above indication, for noumenal tumour, the quantity of expansion, location and lump is clinical definite primary factor in stage.Stage of B cell malignancies patient of determining non-tumour form is complicated more, requires aforesaid serum level to measure.

Be not limited to above-mentioned to the B cell disease and the description in disorderly stage.Can use in diagnosis B cell disease and disorderly field known further feature to determine the stage of patient B cell disease or disorder as standard.

5.5.2 the clinical criteria of diagnosis B cell malignancies

The Case definition of known different B cell malignancies in this area.In history, generally diagnose according to high power amplification form and immune phenotypic combination.Recently, used molecular engineering, for example gene expression profile expand the B cell malignancies the branch sub-definite (referring to, for example: people such as Shaffer, Nature2:920-932 (2002)).Exemplary method to the clinical diagnosis of particular B cell malignancies hereinafter is provided.For a person skilled in the art, other appropriate means is conspicuous.

5.5.2.1 folliculus NHL

Usually, most of NHL (except that the mantle cell lymphoma) have the highly immunoglobulin gene of sudden change, and this is the result of body hypermutation different (SHM).The most of common heredity of NHL is not normal to be the transposition and the sudden change of BCL6 gene.

Folliculus NHL is generally the inertia B cell lymphoma with folliculus growth pattern.It is the second largest common lymphoma in the U.S. and West Europe.Showing this sick intermediate ages is 60 years old, and slight female feature is arranged.Painless lymphadenopathy is modal symptom.Test often shows the blood marrow, also has the participation of peripheral blood sometimes.According to Magnocellular ratio in the folliculus, folliculus NHL is divided into the cytology grade, preponderate continuous divided rank from the folliculus small cleaved cell to maxicell (referring to people such as S.Freedman, Follicular Lymphoma, pp.367-388, InNon-Hodgkin ' s Lymphomas, people such as P.Mauch, eds.LippincottWilliams﹠amp; Wilkins, Philadelphia, PA (2004); People such as T.Lister, FollicularLymphoma, pp.309-324, In Malignant Lymphoma, people such as B.Hancock, eds.Oxford University Press, New York, N.Y. (2000)).

The characteristics of most of folliculus NHL are to cause BCL2 to cross transposition between the karyomit(e) 14 and 18 of expression.The feature of folliculus NHL also be SHM and developmental SHM be similar to secondary nodules (GC) B gene expression of cells distribute (referring to, for example: people such as Shaffer, Nature 2:920-932 (2002)), it is the cell source of generally acknowledging of this malignant tumour.Rearranging of heavy chain and light chain is characteristic feature.The monoclonal surface immumoglobulin of the tumor cells expression of this disease, great majority are expressed IgM.Nearly all folliculus NHL tumour cell is antigen expressed CD19, CD20, CD79a, CD21, CD35 and CD10 all, but lacks the expression of CD5 and CD43.Can in marrow, observe the infiltration of little cleaved cell side girder.(referring to, people such as S.Freedman, Follicular Lymphoma, pp.367-388, In Non-Hodgkin ' sLymphomas, people such as P.Mauch, eds.Lippincott Williams﹠amp; Wilkins, Philadelphia, PA (2004); People such as T.Lister, FollicularLymphoma, pp.309-324, In Malignant Lymphoma, people such as B.Hancock, eds.OxfordUniversity Press, New York, N.Y. (2000)).

The nodular examination of living tissue of cutting off for appraisement organization's structure and cytologic characteristic is depended in the diagnosis of folliculus NHL usually.The aspirate of tiny pin is not enough usually, because the unlikely tissue that can be used to identify that provides of this step can not provide enough tissues to do additional test.Point out that simultaneously bilateral marrow all will carry out examination of living tissue, because content may be inhomogeneous.Additional diagnosis algorithm comprises computerized tomography (CT) scanning of chest x ray, chest, abdomen, neck and pelvis, all blood counting and chemicals distributions.Can utilize flow cytometry and immunohistochemistry to distinguish folliculus NHL and other sophisticated B cell lymphoma.(referring to: people such as S.Freedman, FollicularLymphoma, pp.367-388, In Non-Hodgkin ' s Lymphomas, people such as P.Mauch, eds.Lippincott Williams﹠amp; Wilkins, Philadelphia, PA (2004); People such as T.Lister, Follicular Lymphoma, pp.309-324, In MalignantLymphoma, people such as B.Hancock, eds.Oxford University Press, New York, N.Y. (2000)).

5.5.2.2 mantle cell lymphoma

The mantle cell lymphoma is positioned the mantle segment of secondary hair follicle, and its characteristics are nodular and/or the diffuse growth pattern.Mantle cell lymphoma patient's intermediate ages is 60-65 year, this sick main male sex that infects.For the purpose of diagnosing, the feature that shows usually is generalized lymphadenopathy.In addition, the normal enlargement of the spleen channel.T (11 between this B cell lymphoma and IgH site and the cyclin D1 gene; 14) relevant, it causes crossing of cyclin D1 to be expressed.It is not normal that patient chart more than 50% reveals additional karyomit(e).The mantle cell lymphoma generally is not that feature is (referring to people such as W.Hiddemann, Mantle Cell Lymphoma, pp.461-476, In Non-Hodgkin ' s Lymphomas, people such as P.Mauch, eds.LippincottWilliams﹠amp with SHM; Wilkins, Philadelphia, PA (2004); People such as D.Weisenburger, Mantle Cell Lymphoma, pp.28-41, In Malignant Lymphoma, people such as B.Hancock, eds.Oxford University Press, New York, N.Y. (2000)).

The immunohistochemistry of the immunophenotype of lymphoma mantle cell cell (flow cytometry or frozen section) shows that they are almost always monoclonal, produces surperficial IgM.The lymphoma mantle cell cell also has the surperficial IgD's of generation.Cell expressing antigens c D19, CD20, CD22 and CD24, but do not have CD23.They also express surface antigen CD5, but do not have CD10, it are distinguished out from true property follicular center cell lymphoma, and it is CD5 feminine gender always almost.Usually, find that the outer composition of joint comprises marrow penetrant and liver neoplasm and stomach and intestine nerve tract.The mantle cell lymphoma have an anemia and leukemic performance unrare (referring to people such as A.LaI, Role of Fine NeedleAspiration in Lymphoma, pp.181-220, people such as In W.Finn, eds.Hematopathology in Oncology, Kluwer Academic Publishers, Norwell, MA (2004); People such as W.Hiddemann, Mantle Cell Lymphoma, pp.461-476, In Non-Hodgkin ' s Lymphomas, people such as P.Mauch, eds.LippincottWilliams﹠amp; Wilkins, Philadelphia, PA (2004)).

The diagnosis of mantle cell lymphoma comprises the living tissue inspection of peripheral blood and marrow and lymphoglandula.In addition, cytogenetics research and immunity performance somatotype are useful (referring to people such as W.Hiddemann, Mantle Cell Lymphoma pp.461-476, In Non-Hodgkin ' s Lymphomas in the differentiation diagnosis, people such as P.Mauch, eds.LippincottWilliams﹠amp; Wilkins, Philadelphia, PA (2004); People such as D.Weisenburger, Mantle Cell Lymphoma, pp.28-41, In Malignant Lymphoma, people such as B.Hancock, eds.Oxford University Press, New York, N.Y. (2000)).

5.5.2.3 burkitt's lymphoma

Burkitt's lymphoma is a kind of generally at children and the observed on one's body aggressive B cell lymphoma of Young Adults, and relevant with the major disease of jaw and/or belly usually.About 20% patient has the marrow problem.The region burkitt's lymphoma comprises that Epstein-Barr virus (EBV) malignant cell infects; Divergence form is independent of EBV and infects.Causing the transposition of the c-myc of c-myc gene abnormality to immunoglobulin locus, is that (t (8 for these sick characteristics; 14) (q24; Q32)).What is interesting is, the disappearance of c-myc sequence seems to relate to the divergence form of disease, and the region form generally includes point mutation or insertion (referring to people such as V.Pappa, Molecular Biology, pp.133-157, In Malignant Lymphoma, people such as B.Hancock, eds.OxfordUniversity Press, New York, N.Y. (2000)).Burkitt's lymphoma also is feature with SHM, and malignant cell has the GC of being similar to B gene expression of cells and distribute, and shows that this malignant tumour comes from GC B cell.

The immune phenotype of Burkett's lymphoma shows cell expressing CD19, CD20, CD22 and the CD79a that this is sick, but does not express CD5, CD23, cyclin D or the terminal deoxynucleoside acyl transfer factor of changeing.Usually, these cells are positive to CD10 and BCL6, and usually BCL2 are negative (referring to people such as I.Magrath, Burkitt ' s Lymphoma, pp.477-501, InNon-Hodgkin ' s Lymphomas, people such as P.Mauch, eds.LippincottWilliams﹠amp; Wilkins, Philadelphia, PA (2004)).

High-grade B cell Bai Jiteshi sample lymphoma is the lymphoma boundary line between burkitt's lymphoma and the big B cell lymphoma.This lymphadenomatous cell expressing CD19 and CD20, but the expression of the CD10 that almost always exists in true property burkitt's lymphoma does not but usually exist.Because this and other feature, some people believes that this lymphoma should be classified as the diffusion large B cell lymphoid tumor (referring to K.Maclennan, Diffuse Aggressive B cell Lymphoma, pp.49-54, In Malignant Lymphoma, people such as B.Hancock, eds.Oxford University Press, New York, N.Y. (2000)).

The detection of the transposition relevant with this lymphoma is depended in the diagnosis of burkitt's lymphoma usually; Therefore, carry out traditional cytogenetics analysis usually.Use length to be connected with the Ig-myc that fluorescence in situ hybridization (FISH) detects at the transposition relevant with this disease and other gene alteration place apart from polymerase chain reaction technology.(referring to, R.Siebert waits the people, Blood 91:984-990 (1998); T.Denyssevych waits the people, Leukemia, 16:276-283 (2002)).

5.5.2.4 the large B cell lymphoid tumor (DLBCL) of diffusion

DLBCL is modal non-Hodgkin lymphomas, and can be produced by small B-cell lymphoma, follicular lymphoma or marginal zone lymphoma.In general, the patient shows lymphadenopathy; Yet the patient of significant proportion also shows the outer site of joint, relates to the most common of stomach and intestine.Observe relate to marrow be approximately the patient 15% (referring to people such as Armitage, Diffuse LargeB cell Lymphoma, pp.427-453, In Non-Hodgkin ' s Lymphomas, people such as P.Mauch, eds.Lippincott Williams﹠amp; Wilkins, Philadelphia, PA (2004)).The heterogeneity of clinical, biology and morphological specificity makes this group lymphoma be difficult to carry out subclassification.Yet two different subgroups have confirmed that one is the expressing gene of feature with embryonic center B cell (GC-DLBCL), and another crosses the gene of expressing in peripheral blood B cell.GC-DLBCL patient's survival rate significantly is better than the patient of activating B cell type (ABC)-DLBCL.(referring to: W.Chan, Archives of Pathology and Laboratory Medicine128 (12): 1379-1384 (2004)).

DLBCL express cell surface antigen CD19, CD20, CD22 and CD79a.All express CD10 in most of cases, express CD5 in about 10% the case (referring to K.Maclennan, Diffuse Aggressive B cell Lymphoma, pp.49-54, In Malignant Lymphoma, people such as B.Hancock, eds.Oxford University Press, New York, N.Y. (2000)).It is mark that DLBCL often translocates to the IgH site with the abnormality of BCL6 and/or BCL2.The characteristics of GC B cell sample (GC) DLBCL be to have highly sudden change immunoglobulin gene SHM and have developmental SHM in the malignant clone of GC B cell sample gene expression profile.Most GC DLBCL lives through the change of immunoglobulin class.The characteristics of ABC-DLBCL are the high level expression of NF-κ B target gene, comprise BCL2, interferon regulatory factor 4, CD44, FLIP and cyclin D.Have SHM, and do not have developmental SHM, and ABC-DLBCL does not have the distribution of GC B cellular gene expression.Nearly all ABC-DLBCL expresses IgM high-levelly.

5.5.2.5 tubercle outer edge area lymphoma

Tubercle outer edge area lymphoma is the tubercle perilymph knurl in a kind of adenoid organ (for example: stomach, sialisterium, lung and Tiroidina) that is present in common inorganizable property.It is a kind of main infection the elderly's a disease, and the mean age of infection was above 60 years old.Usually, before the lymphoma development chronic inflammatory diseases or self-immunprocess appear.Stomach mucous membrane-associated lymphoid tissue (MALT) lymphoma, the modal kind of marginal zone lymphoma, relevant with helicobacter pylori infection.Studies show that after the antibiotic administration and can eliminate symptom by the eradicate helicobacter pylori infection.The symptom that gastric MALT lymphoma shows comprises nonspecific maldigestion, pained, nauseating, gastrointestinal hemorrhage and anaemia.The general symptom is rarely found, as the raising of lactic dehydrogenase enzyme level.(referring to: people such as J.Yahalom, Extranodal Marginal Zone B cell Lymphoma of Mucosa-AssociatedLymphoid Tissue, pp.345-360, In Non-Hodgkin ' s Lymphomas, people such as P.Mauch, eds.Lippincott Williams﹠amp; Wilkins, Philadelphia, PA (2004); J.Radford, Other Low-Grade Non-Hodgkin ' s Lymphomas, pp.325-330, InMalignant Lymphoma, people such as B.Hancock, eds.Oxford University Press, New York, N.Y. (2000).General B symptom comprise surpass 2 week fever surpass 38 ℃, do not have infection, night sweat, extreme fatigue or surpassing 10% the sign that loses weight without reason in 6 months time more than or equal to body weight before).

The characteristics of the lymphadenomatous immunophenotype of MALT are the expression that lacks of the expression of CD20, CD79a, CD21 and CD35 and CD5, CD23 and CD1O.Only about half of MALT lymphoma is expressed CD43.General immunoglobulin (Ig) of expressing in the tumour cell of this disease is IgM, and does not express IgD.These features are to distinguish this lymphoma and other little B cell lymphoma, for example key of lymphoma mantle cell, lymphocytic lymphoma and follicular lymphoma.In 60% MALT lymphoma case, trisome 3 types have been reported.In the MALT lymphoma of the stomach of 25-40% and lung, observed t (11; 18).In other MALT lymphoma, still less observe this transposition.T (11; 18) relevant with the nuclear expression of BCL10.(referring to: people such as J.Yahalom, Extranodal MarginalZone B cell Lymphoma of Mucosa-Associated Lymphoid Tissue, pp.345-360, In Non-Hodgkin ' s Lymphomas, people such as P.Mauch, eds.LippincottWilliams﹠amp; Wilkins, Philadelphia, PA (2004)).Marginal zone lymphoma is a feature with SHM and developmental SHM usually.

Diagnosis algorithm comprises the identity of measuring cell surface marker with immunophenotyping or fluidic cell method.In addition, should use molecular genetic assay determination t (11; 18) existence because this be this disease can be to the indicator of microbiotic reaction.Histology can be used for determining the existence of helicobacter pylori.Additional test should comprise complete blood count, basic biochemical test, and it comprises the test to serum lactic dehydrogenase; Abdominal CT scan, chest and pelvis and BMB.(referring to: people such as J.Yahalom, Extranodal Marginal Zone B cell Lymphoma of Mucosa-Associated Lymphoid Tissue, pp.345-360, In Non-Hodgkin ' s Lymphomas, people such as P.Mauch, eds.Lippincott Williams﹠amp; Wilkins, Philadelphia, PA (2004)).

5.5.2.6 the B cell lymphoma of tubercle marginarium

The B cell lymphoma of tubercle marginarium is a kind of lymphoma of new relatively classification, to it disclosure seldom.It is the tubercle B cell lymphoma at a kind of initial stage, and outer and splenic marginal zone lymphoma has common heredity and morphological specificity with tubercle, but no fix is outside spleen or tubercle.Reported that hepatitis C virus is relevant with this lymphoma, for example:

Syndrome.(referring to: people such as F.Berger, Nodal Marginal Zone B cell Lymphoma, pp.361-365, In Non-Hodgkin ' sLymphomas, people such as P.Mauch, eds.Lippincott Williams﹠amp; Wilkins, Philadelphia, PA (2004)).

The lymphoma of tubercle marginarium has heterogenetic cytology and morphology.Because the maxicell of its higher proportion, this lymphoma are different from other marginal zone lymphoma (spleen outer with tubercle), can not classify as the low B cell lymphoma that waits of true property to it.The phenotype of the heredity of tubercle marginal zone lymphoma and immunity comprises the expression of CD19, CD20, BCL2, sIgM and tenuigenin IgG (cIg).These cells are not expressed CD5, CD1O, CD23, CD43 or cyclin D1.The lymphadenomatous transposition feature of MALT, t (11; 18), in the tubercle marginal zone lymphoma, do not observe.(referring to: people such as F.Berger, Nodal MarginalZoneBcell Lymphoma, pp.361-365, In Non-Hodgkin ' s Lymphomas, people such as P.Mauch, eds.LippincottWilliams﹠amp; Wilkins, Philadelphia, PA (2004)).

5.5.2.7 splenic marginal zone lymphoma

The splenic marginal zone lymphoma is a kind of lesser tubercle B cell lymphoma of slower development, and it shows the Clinical symptoms that significant splenomegaly and peripheral blood and marrow are infiltrated.In addition, report has the liver damage of higher level.The effect of hepatitis C virus is this lymphadenomatous prerequisite.The lymphadenomatous immunophenotype of splenic marginal zone is generally CD20 ⁺, IgD ⁺, BCL2 ⁺, p27 ⁺, CD3 ^-, CD5 ^-, CD10 ^-, CD23 ^-, CD38 ^-, CD43 ^-, BCL-6 ^-And cyclin D1 ^-Hereditary feature comprises that 7q disappearance, p53 change and SHM.(referring to: people such as M.Piris, Splenic Marginal ZoneLymphoma, pp.275-282, In Non-Hodgkin ' s Lymphomas, people such as P.Mauch, eds.Lippincott Williams﹠amp; Wilkins, Philadelphia, PA (2004)).

The mensuration of immunophenotype pair cell surface markers identity is depended in diagnosis usually.Heredity and biochemical analysis combine with the data of cell surface marker, help to distinguish this lymphoma and other little B cell lymphoma.(referring to: people such as M.Piris, Splenic Marginal Zone Lymphoma, pp.275-282, In Non-Hodgkin ' s Lymphomas, people such as P.Mauch, eds.LippincottWilliams﹠amp; Wilkins, Philadelphia, PA (2004)).

5.5.2.8 acute (B cell) lymphocytic leukemia (ALL)

ALL is a kind of tumour based on marrow, and its main infected children is the highest in 1-5 year intercurrent disease rate.The common sympton that shows comprises fatigue, lethargic sleep, fever and bone and arthralgia.Tired relevant with the severity of anemia that shows with lethargic sleep.The increase of white blood cell count is common performance.Chest is according to often demonstrating the skeleton pathology.Common jaw is expanded outward, and it influences central nervous system, spermary, lymphoglandula, liver, spleen and kidney.Only approximately can observe the anterior mediastinum piece in the case of making a definite diagnosis recently of 5-10%.(referring to: people such as J.Whitlock, Acute Lymphocytic Leukemia, pp.2241-2271, In Wintrobe ' s Clinical Hematology, Tenth Edition, people such as G.Lee, eds.Williams﹠amp; Wilkins, Baltimore, MD (1999)).

The immunophenotype of ALL is CD1O ⁺, CD19 ⁺, CD20 ⁺And CD24 ⁺Pre B cell ALL cell expressing tenuigenin, rather than surface immumoglobulin, and mature B cell ALL (it accounts for the 1-2% of ALL case) can distinguish by the expression of surface immumoglobulin mutually with the leukemia of other B clone.The cytogenetics feature of ALL comprises t (8; 14), t (2; 8) and t (8; 22).Though it seldom detects in the cytogenetics level, t (12; 21) may be with the Childhood ALL relevant modal cytogenetics not normal (in about 25% case, observing).(referring to: people such as M.Kinney, Classification and Differentiation of the AcuteLeukemias, pp.2209-2240, In Wintrobe ' s Clinical Hematology, TenthEdition, people such as G.Lee, eds.Williams﹠amp; Wilkins, Baltimore, MD (1999); People such as J Whitlock, Acute Lymphocytic Leukemia, pp.2241-2271; InWintrobe ' s Clinical Hematology, Tenth Edition, people such as G.Lee, eds.Williams﹠amp; Wilkins, Baltimore, MD (1999)).

Bone extract and examination of living tissue are depended in the accurate diagnosis of acute leukemia usually.The extract smear is used for morphology, immunology and cytological mensuration.Lymphoblastic checking in the marrow is the diagnosis of ALL.Exist in the marrow and can make a definite diagnosis ALL greater than 5% leukemia lymphocytoblast, but most the requirement greater than 25% to obtain sure diagnosis.Lumbar puncture is used to diagnose central nervous system damage.In ALL, can find the raising of serum uric acid level and serum lactic dehydrogenase (SLDH) level.(referring to: people such as M.Kinney, Classification and Differentiation of the AcuteLeukemias, pp.2209-2240, In Wintrobe ' s Clinical Hematology, TenthEdition, people such as G.Lee, eds.Williams﹠amp; Wilkins, Baltimore, MD (1999); People such as J.Whitlock, Acute Lymphocytic Leukemia, pp.2241-2271; InWintrobe ' s Clinical Hematology, Tenth Edition, people such as G.Lee, eds.Williams﹠amp; Wilkins, Baltimore, MD (1999)).

5.5.2.9 lymphocytic leukemia (CLL)/little B cell lymphocyte lymphoma (SLL)

CLL/SLL is leukemic frequent species.When this disease enters peripheral blood and marrow, be called CLL.Yet, when lymphoglandula and other tissue by with CLL in cell identical cell infiltration on immunity and morphology, but when this disease lacked leukemic feature, this disease was called as SLL.This disease mainly infects the elderly, and the male sex's sickness rate is than women height.Painless lymphadenopathy is modal performance.Hypogammaglobulinaemia is to show all immunoglobulin (Ig)s, rather than the common sympton of most of cases of the CLL/SLL of the level of the specific subclass of any immunoglobulin (Ig) reduction.In the conventional blood counting common asymptomatic patient (the lymphocyte value surpasses 5000 * 10 ⁹/ L).Nearly 20% CLL/SLL case has been reported the B symptom.Additional diagnostic characteristic is that marrow has and surpasses 30% and infiltrated by jejune lymphocyte.Biopsy of lymph node shows to have usually easily distinguishes lymphocytic nodular infiltration.Autoimmune phenomena is often relevant with CLL/SLL, and it comprises autoimmune hemolytic anemia and immune thrombopenia.(referring to: people such as J.Gribben, Small B cell Lymphocytic Lymphoma/Chronic LymphocyticLeukemia and Prolymphocyte Leukemia, pp.243-261, Ln Non-Hodgkin ' sLymphomas, people such as P.Mauch, eds.Lippincott Williams﹠amp; Wilkins, Philadelphia, PA (2004); K.Maclennan, Diffuse Indolent B cellNeoplasms, pp.43-47, In Malignant Lymphoma, people such as B.Hancock, eds.Oxford University Press, New York, N.Y. (2000); Clinical Oncology, people such as A.Neal, Neal, Hoskin and Oxford University Press, co-publ.NewYork, NY (2003)).

Compare with many low B cell malignancies that wait, seldom in CLL/SLL, find nonrandom mutual transposition.But, the cytogenetic not normal disappearance that comprises 13q14,11q22-23 and 17q13 place of other of report, both relate to the p53 site back.About 20% patient chart reveals trisome 12 types.The higher level that the high level of β-2 microglobulin, CD38 express and the generation of tumor necrosis factor-alpha are whole features of CLL/SLL.The immunophenotype of CLL/SLL is helpful to diagnosing, and it comprises the faint expression of surface immumoglobulin and the expression of cell antigen CD19, CD20 and common CD5 and CD23, and described surface immumoglobulin is often referred to IgM, or IgM and IgG.(referring to: people such as J.Gribben, Small B cell Lymphocytic Lymphoma/Chronic LymphocyticLeukemia and Prolymphocyte Leukemia, pp.243-261, In Non-Hodgkin ' sLymphomas, people such as P.Mauch, eds.Lippincott Williams﹠amp; Wilkins, Philadelphia, PA (2004); K.Maclennan, Diffuse Indolent B cellNeoplasms, pp.43-47, In Malignant Lymphoma, people such as B.Hancock, eds.Oxford University Press, New York, N.Y. (2000)).

5.5.2.10B cell prolymphocyte leukemia (PLL)

PLL once was considered to the variant of CLL, and it is now know that, and it is a kind of visibly different disease.PLL is the disease of elderly men normally, it is characterized in that very high white blood cell count is (greater than 200 * 10 ⁹/ L) and splenomegaly.Other symptom comprises anaemia and thrombopenia.Prolymphocyte among the PLL comprises and surpasses 55% blood and the cell in the marrow.Compare with CLL, in PLL, seldom observe autoimmune phenomena.(referring to: people such as J.Gribben, Small B cell LymphocyticLymphoma/Chronic Lymphocytic Leukemia and ProlymphocyteLeukemia, pp.243-261, In Non-Hodgkin ' s Lymphomas, people such as P.Mauch, eds.Lippincott Williams﹠amp; Wilkins, Philadelphia, PA (2004)).

The immunophenotype of PLL is characterised in that the expression of CD19, CD21, CD22, CD24 and FMC7.The cell of PLL is not expressed CD23, and great majority are not expressed CD5.It is not normal that the PLL cell shows complicated karyomit(e), more modal disappearance with 13q14 and 11q23.The pattern of p53 sudden change is with observed different in CLL in the PLL cell.Complete blood count, histological, immunophenotype and analysis heredity are depended in differential diagnosis usually.(referring to: people such as J.Gribben, Small B cell Lymphocytic Lymphoma/Chronic Lymphocytic Leukemiaand Prolymphocytic Leukemia, pp.243-261, In Non-Hodgkin ' sLymphomas, people such as P.Mauch, eds.Lippincott Williams﹠amp; Wilkins, Philadelphia, PA (2004)).

5.5.2.11 hairy cell leukemia (HCL)

HCL is a kind of chronic leukemia of rare, slower development, and the male sex is than women's easy infection more, and mainly infects the middle-aged people.Classical symptom comprises a large amount of splenomegaly and pancytopenia.Peripheral blood and marrow comprise typically " hair cell ", and it is bone-marrow-derived lymphocyte by the tenuigenin projection.HCL patient above 90% marrow occurs and infiltrates.(referring to: Clinical Oncology, people such as A.Neal, Neal, Hoskin and Oxford University Press, co-publ.NewYork, NY (2003); J.Johnston, Hairy Cell Leukemia, pp.2428-2446, In Wintrobe ' s Clinical Hematology, Tenth Edition, people such as G.Lee, eds.Williams﹠amp; Wilkins, Baltimore, MD (1999)).

The cytogenetics analysis revealed exists pure lines not normal in 19% case, and relates to karyomit(e) 5,7 and 14 quantitatively with structural not normal.In the hairy cell leukemia, the serum level of TNF-α improves, and it is relevant with the lump load.Hairy cell leukemia cell expressing surface immumoglobulin (IgG and IgM) and CD11c, CD19, CD20, CD22 and typically express CD25.In addition, also express FMC7, HC-2 and CD103.The HCL cell is not expressed CD5 or CD1O.Diagnosis is usually directed to the use of marrow extract, cytogenetics, blood smear and immunophenotype.(referring to: Clinical Oncology, people such as A.Neal, Neal, Hoskin and Oxford UniversityPress, co-publ.New York, NY (2003); J.Johnston, Hairy Cell Leukemia, pp.2428-2446, In Wintrobe ' s Clinical Hematology, Tenth Edition, people such as G.Lee, eds.Williams﹠amp; Wilkins, Baltimore, MD (1999)).

5.5.2.12 the acute lymphocytoblast leukemia of precursor B cell lymphocytoblast lymphoma/pre-B cell/lymphocytoblast lymphoma

The acute lymphocytoblast leukemia of precursor B cell lymphocytoblast lymphoma/pre-B cell/the lymphocytoblast lymphoma is the disease of a kind of precursor T or B cell.T and B cell lymphoblastic lymphoma homomorphosis, but may cause clinically difference according to the degree that marrow infiltrates or marrow relates to.The lymphoblastic lymphoma of 85-90% is that the T cell is by the cell-derived resistates of T institute deutero-.Average (infection) age of lymphoblastic lymphoma is 20 years old, and the male sex is in the majority.The infringement of periphery lymphoglandula is the common feature that shows, and especially can betide neck, clavicle and arm-pit areas.This disease often shows bone marrow damage.The performance of central nervous system is uncommon, but often appears in the recurrent infection case.Other position of relating to can comprise that liver, spleen, bone, skin, pharynx and spermary are (referring to people such as J.Sweetenham, Precursor B-and T-CeIlLymphoblastic Lymphoma, pp.503-513, In Non-Hodgkin ' s Lymphomas, people such as P.Mauch, eds.Lippincott Williams﹠amp; Wilkins, Philadelphia, PA (2004)).

Precursor B cell lymphoblastic lymphoma is expressed jejune mark B cell marking for example CD99, CD34 and terminal deoxyribotide transferase.These cells are also expressed CD79a, CD19, express CD20 sometimes, and generally lack the expression of CD45 and surface immumoglobulin.At 11q23 and t (9; 22) (q34; Q11.2) and t (12; 21) (p13; Q22) transposition of locating is relevant with bad prognosis.Good prognosis is relevant with the hyperdiploid karyotype, especially with trisome 4,10 and 17 types and t (12; 21) (p13; Q22) relevant.(referring to: people such as J.Sweetenham, Precursor B-and T-CeIl Lymphoblastic Lymphoma, pp.503-513, InNon-Hodgkin ' s Lymphomas, people such as P.Mauch, eds.LippincottWilliams﹠amp; Wilkins, Philadelphia, PA (2004)).

Diagnostic test comprises biopsy of lymph node, blood test, x ray, CT scan and lumbar puncture, to measure the cerebrospinal fluid of malignant cell.

5.5.2.13 the mediastinum large B cell lymphoid tumor at initial stage

The mediastinum large B cell lymphoid tumor at initial stage is a kind of diffustivity large B cell lymphoid tumor that mainly betides in the young woman, it is characterized by the anterior mediastinum lump of the part intrusion that comes from thymus gland.Can be diffused into not end node farthest, and bone marrow damage is uncommon.Common general symptom.This disease and nodular large celllymphoma are similar, but it has different heredity, immunity and morphological specificity.

The tumour cell immunophenotype of the mediastinum large B cell lymphoid tumor at initial stage often is the surface immumoglobulin feminine gender, but expresses this B cell conjugated antigen, as: CD19, CD20, CD22 and CD79a.Also express CD1O and BCL6 usually.Plasmocyte bonding mark CD15, CD30, epithelial membrane antigen (EMA) are seldom expressed.BCL6 and c-myc sequence in the gene are uncommon yet.The super sudden change of immunoglobulin (Ig) reorganization, immune globulin variable region and the gene of pure lines follows the existence of the super sudden change of BCL6 to illustrate that this lymphoma is derived from sophisticated germinal center or the back B of germinal center cell.Chromosome translocation that may be relevant with the tumour of this disease is to observed similar in other form of diffustivity large celllymphoma.(referring to: people such as P.Zinzani, Primary MediastinalLarge B cell Lymphoma, pp.455-460, In Non-Hodgkin ' s Lymphomas, people such as P.Mauch, eds.Lippincott Williams﹠amp; Wilkins, Philadelphia, PA (2004)).

The diagnostic test of the mediastinum large B cell lymphoid tumor at initial stage generally includes comprehensive physical examination, blood and biochemical analysis, whole body computer planigraphy and BMB completely.Gallium-67 scanning be to by stages, treatment replys and recurs the efficiency test of evaluation and test.(referring to: people such as P.Zinzani, Primary Mediastinal Large B cell Lymphoma, pp.455-460, In Non-Hodgkin ' s Lymphomas, people such as P.Mauch, eds.LippincottWilliams﹠amp; Wilkins, Philadelphia, PA (2004)).

5.5.2.14 lymph-plasma cell lymphoma (LPL)/lymph-plasma cellular immunization cytoma/WaldstromShi macroglobulinemia

LPL/ lymph-plasma cellular immunization cytoma/WaldstromShi macroglobulinemia is a kind of common slower development, and often invades the tubercle lymphoma of marrow, lymphoglandula and spleen.Its normally a kind of geriatric disease, the male sex slightly accounts for leading.Most patients in its serum, have monoclonal IgM paraprotein (＞3g/dL), it causes the too high viscosity of serum.Tumour cell has the cytoplasm form.The characteristics of LPL hypotype are the transposition that repeats between karyomit(e) 9 and 14, and it relates to PAX5 and immunoglobulin heavy chain locus.The characteristics of LPL are SHM and developmental SHM, and it is considered to be derived from back-GC B cell.(referring to people such as A.Rohatiner, LymphoplasmacyticLymphoma and

Macroglobulinemia, pp.263-273, In Non-Hodgkin ' s Lymphomas, people such as P.Mauch, eds.LippincottWilliams﹠amp; Wilkins, Philadelphia, PA (2004); K.Maclennan, DiffuseIndolent B cell Neoplasms, pp.43-47, In Malignant Lymphoma, B.Hancock waits the people, eds.Oxford University Press, New York, N.Y. (2000); People such as A.LaI, Role of Fine Needle Aspiration in Lymphoma, pp.181-220, people such as InW.Finn, eds.Hematopathology in Oncology, Kluwer AcademicPublishers, Norwell, MA (2004)).

The immunophenotype of this disease shows the expression of B cell conjugated antigen CD19, CD20, CD22 and CD79a, and lacks the expression of CD5, CD10 and CD23.Abundant surface immumoglobulin and the existence of CD20, the shortage that CD5 and CD23 express, and CIg existence is the feature that helps to distinguish this disease and chronic lymphocytic leukemia.The symptom of this disease also is t (9; 14) (p13; Q32).(referring to: people such as A.Rohatiner, Lymphoplasmacytic Lymphomaand

Macroglobulinemia, pp.263-273, In Non-Hodgkin ' sLymphomas, people such as P.Mauch, eds.Lippincott Williams﹠amp; Wilkins, Philadelphia, PA (2004); K.Maclennan, Diffuse Indolent B cellNeoplasms, pp.43-47, In Malignant Lymphoma, people such as B.Hancock, eds.Oxford University Press, New York, N.Y. (2000); People such as R.Chaganti, Cytogenetics of Lymphoma, pp.809-824, In Non-Hodgkin ' s Lymphomas, people such as P.Mauch, eds.Lippincott Williams﹠amp; Wilkins, Philadelphia, PA (2004)).

Deagnostic test generally comprises complete blood count, kidney and liver function test, CT scan, examination of living tissue and marrow extraction, protein electrophoresis, measures the quantity and the sign of paraprotein and serum viscosity.The measurement of β 2-microglobulin is used as the prognosis inspection.(referring to: people such as A.Rohatiner, Lymphoplasmacytic Lymphoma and

Macroglobulinemia, pp.263-273, In Non-Hodgkin ' s Lymphomas, people such as P.Mauch, eds.LippincottWilliams﹠amp; Wilkins, Philadelphia, PA (2004)).

5.5.2.15 non-acute lymphoblastic leukemia

Non-acute lymphoblastic leukemia is the hypotype of a kind of ALL, and it lacks B-or T-cell characteristic.The paotoblastic phenotype analytical of leukemia shows a kind of typical non-ALL pattern, that is: CD10 (common ALL antigen)-feminine gender, the HLA-DR-positive, and CD19 consumingly (the B4)-positive (referring to: people such as Katz (1988) Blood71 (5): 1438-47).

5.5.2.16 hodgkin's lymphomas

Hodgkin's lymphomas usually occurs in the young adult lymphoglandula.It can be divided into typical hypotype and rare trifle lymphocyte is main hypotype.Typical type performance SHM, but do not show developmental SHM, and do not have GC B cellular gene expression to distribute.On the contrary, the trifle lymphocyte is that the characteristics of main hypotype are that SHM and developmental SHM and GC B cellular gene expression distribute.These two types clinically with biologically different, but they have some common trait, for example: lack tumour cell in optimum inflammatory cells.(people such as B.Schnitzer, HodgkinLymphoma, pp.259-290, In W.Finn and L.Peterson, eds.Hematopathologyin Oncology, Kluwer Academic Publishers, Norwell, MA (2004)).

The most common on the phenotype is characterized as painless lymphoglandula amplification, usually at neck, but occasionally at inguinal region.The wax of lymphoglandula and pale asphyxia also are the features of this disease.In about 1/3rd patient, observe the B symptom.Independent tubercle is outer to be damaged seldom, and it approximately is the 10-20% of its number that observed diffusion occurs in the outer case of tubercle.(referring to: people such as P.Johnson, Hodgkin ' s Disease:Clinical Features, pp.181-204, In MalignantLymphoma, people such as B.Hancock, eds.Oxford University Press, New York, N.Y. (2000)).

Reed-Sternberg (RS) cell is the malignant cell of hodgkin's lymphomas.RS cell and its variant are expressed CD15, CD25, CD30 and TfR.The polyclonal tenuigenin immunoglobulin (Ig) of these cell expressings in addition.In most of cases of hodgkin's lymphomas, the RS cell is not expressed CD45, and this is the feature that this disease and non-Hodgkin lymphomas are distinguished in a help.Proved that in only about half of hodgkin's lymphomas case, Epstein Barr virus is present in the Reed-Sternberg cell, but its effect is not clear.

The most normal use biopsy of lymph node diagnoses.Other deagnostic test comprises full blood count, and (usually visible hematology test is normal; In about 20% case, white blood cell count is less than 1.0 * 10 ⁹/ L), erythrocyte sedimentation rate (raising), biochemical test through the commitment of this disease of being everlasting, it comprise electrolytic solution, urea, creatinine, urate, calcium (hypercalcemia is rarely found, but just relevant with bone infringement on a large scale when occurring), liver blood test, serum lactic dehydrogenase (level raise normal with early stage disease relevant), albumin and beta ₂-microglobulin (β 2-M).The CT scan of Lymphanigiograms and chest x ray and chest, belly and pelvis is very important to the scope of confirming the outer infringement of not normal lymphoglandula and tubercle.Undesired when bone marrow damage, and this bioptic result it is generally acknowledged and can select to carry out BMB when not influencing clinical arrangement or prognosis.Usually do not carry out splenectomy now, because it seldom influences arrangement, and CT or MRI image provide the message of relevant spleen situation.Significantly improving of p55, TNF and sICAM-1 level is relevant with the complete reaction rate with the appearance of stage of this disease, symptom.(referring to: people such as P.Johnson, Hodgkin ' s Disease:Clinical Features, pp.181-204, In MalignantLymphoma, people such as B.Hancock, eds.Oxford University Press, New York, N.Y. (2000); Clinical Oncology, people such as A.Neal, Neal, Hoskin and OxfordUniversity Press, co-publ.New York, NY (2003); R.Stein, Hodgkin ' sDisease, pp.2538-2571, In Wintrobe ' s Clinical Hematology, TenthEdition, people such as G.Lee, eds.Williams﹠amp; Wilkins, Baltimore, MD (1999)).

5.5.2.17 multiple myeloma

Multiple myeloma is a kind of malignant tumour of plasma cell.Tumour cell is positioned at marrow, and the infringement of the bone of molten bone is a feature.At one of immunoglobulin locus and various other genes, as the chromosome translocation of the exchange between cyclin D1, cyclin D3, cMAF, MMSET (multiple myeloma SET-functional zone albumen) or the fibroblast growth factor receptor3, be considered to the carcinogenic factor at initial stage.The characteristics of multiple myeloma are SHM5, and the cells of origin of generally acknowledging is a back-GCB cell.Multiple myeloma is generally at first passed through symptom, for example superinfection, fatigue, pain and kidney problems and obtain identifying, and by clinical trial checking (referring to, for example: Cancer:Principles and Practice of Oncology.6th edition.DeVita, V.T.Hellman, S. and Rosenberg, S.A.editors.2001 LippincortWilliams and Wilkins Philadelphia, PA 19106pp.2465-2499.)

In certain embodiments, the candidate patient who accepts the compositions and methods of the invention treatment can be through the further deagnostic test to blood and/or urine, to confirm the diagnosis or the suspection of multiple myeloma, include but are not limited to, determine by complete blood count (CBC) test whether the cell category of reporting is in its normal range in CBC, it is known in the art, distribute to determine the level of various blood constitutents, for example albumin by hematochemistry, blood urea nitrogen (BUN), calcium, whether creatinine and serum lactic dehydrogenase (LDH) depart from standard value.Also can detect beta ₂-microglobulin (β ₂-M) serum level, and make mark substitute IL-6, a kind of somatomedin of myeloma cell.Urinalysis can be used for measuring the protein level in the urine.Electrophoresis can be used for measuring various proteic levels, comprises the M albumen in blood (being called serum protein electrophoresis or SPEP) or the urine (being called urine electrophoresis or UEP).A kind of additional test is called as immunofixation electrophoresis (IFE) or immunoelectrophoresis, also can be used to provide more relevant customizing messages that show the albumen type of not normal antibody.Various proteic mensuration change and ratio, especially M albumen, can be used for following the trail of the myelomatosis advancing of disease, and generation is replied to treatment plan.The characteristics of multiple myeloma are that the M of myelomatosis tumor cell secretion is proteic and roll up.

Also can be used to confirm the diagnosis or the suspection of multiple myeloma for the deagnostic test of bone, comprise, but be not limited to, X ray and other image measurement--comprise bone (skeleton) inspection, magnetic resonance imaging MRI (MRI) and computed axial tomography (CAT), are also referred to as computed tomography analysis (CT)--variation that can measure skeleton and number and the size of measuring tumour in the bone.Marrow extraction or BMB can be used for detecting the quantity increase of marrow mesoplasmatocyte.Extraction requires the bone marrow fluid sample, and examination of living tissue requires the solid bone tissue sample.In two tests, preferably take from the sample of pelvis (hipbone).Breastbone (breastbone) also can be used for the extraction of marrow.

Multiple myeloma patients generally is divided into following three classes, and this can help to determine effective treatment plan.The general feature of the MG (MGUS) that feature is not clear is a serum M protein level less than the plasmocyte of 3g/dL, marrow pure lines less than 10%, the evidence that does not have other B cell disorder, and relevant organ or tissue injury, for example hypercalcemia (serum calcium level rising), the serum creatinine by increasing, anaemia or bone damage the renal failure that shows.Asymptomatic myelomatosis generally is a Phase I, and comprises the multiple myeloma (IMM) of smouldering property multiple myeloma (SMM) and slower development.The characteristics of SMM are serum M protein more than or equal to 3g/dL, and the characteristics of IMM are that the plasmocyte of marrow pure lines is more than or equal to 10% of medullary cell.The characteristics of Symptomatic myelomatosis are the M albumen in serum and/or the urine, the characteristics that comprise the multiple myeloma of Phase are the plasmocyte of marrow pure lines or the appearance of plasmoma, and the characteristics of Phase I multiple myeloma are the defective of relevant organ or tissue.

The osteosclerosis myelomatosis is the part (polyneuropathy, organomegaly, incretopathy monoclonal gammopathy and skin lesion) of rare POEMS syndromes.The sickness rate peak value is that the age was at 40 to 50 years old.The general symptom comprises that pathology, marrow plasmocyte＜5%, normal CBC, the thrombocyte of bone increase and the organomegaly.CSF has the high protein of acellular performance.M-protein level low (＜3g/dL, intermediate value=1.1g/dL); Heavy chain class-be generally α or γ; Light chain class-be generally λ; Rare urine mono-clonal and cryoglobulinemia once in a while.The weak neuropathy of nearside and distally appears in 50% patient, and unconsciousness is more serious in robust fibre than in fine-fibered; And demyelination is hidden with long-term distally.

Smoulder multiple myeloma patients and show the stable disease of several months/several years usually; There are not anaemia, bone pathology, renal insufficiency or hypercalcemia; Have in marrow and the mono-clonal serum protein＞10% plasmocyte.The judgement of smouldering multiple myeloma is consistent with the diagnosis of multiple myeloma; Yet, do not have the evidence of process.These are cases of slower development, and the tumour cell lump of diagnosis is few, the plasmacytic ratio of marrow low (＜0.5%) of S position phase.Specific Clinical symptoms comprises: serum M protein level＞3g/dL and/or marrow plasmocyte 〉=10%; There are not anaemia, renal failure, hypercalcemia, the infringement of dissolved bone.

Slower development (or asymptomatic) multiple myeloma is a kind of not reveal any symptoms, usually the accidental multiple myeloma of making a definite diagnosis after the research of screening experiment chamber.The multiple myeloma of slower development is similar to smouldering property myelomatosis, but bone pathology and anemia are seldom arranged.Most of cases of the multiple myeloma of slower development developed into tangible multiple myeloma in 3 years.The standard of diagnosis is identical with multiple myeloma, except: there are not bone pathology or asymptomatic solvability pathology (X-ray examination); For IgG, M composition level＜3g/dL, IgA2g/dL, urine light chain＜4g/24h; Oxyphorase＞10g/dL, serum calcium are normal, serum creatinine＜2mg/dL and do not have infection.

5.5.2.18 solitary plasmacytoma

Solitary plasmacytoma is one of scope of plasma cell tumor, its scope from the benign monoclonal gammopathy to the solitary plasmacytoma to multiple myeloma.All about 70% of the solitary plasmacytoma case finally can cause multiple spinal cord knurl.The characteristics of these diseases are to produce the propagation of the B cell of characteristic Paraprotein.The plasmocyte that solitary plasmacytoma causes being sheerly is bred on isolated site, the outer tissue site of normally single bone or jaw.The Case definition of solitary plasmacytoma comprises single pathology, normal bone examination of living tissue, negative skeleton inspection, no anaemia, normal calcium and the renal function that confirms on the histology.The minimum that most of patient charts reveal serum M albumen (paraprotein) increases.The median ages of diagnosis is 50-55, approximately than the young 5-10 of the median ages of multiple spinal cord knurl.(referring to: C.Wilson, The Plasma Cell Dycrasias, pp.113-144, In W.Finn and L.Peterson, eds.Hematopathology inOncology, Kluwer Academic Publishers, Norwell, MA (2004), people such as S.Chaganti, Cytogenetics of Lymphoma, pp.809-824, In Non-Hodgkin ' sLymphomas, people such as P.Mauch, eds.Lippincott Williams﹠amp; Wilkins, Philadelphia, PA, (2004)).

It is similar with multiple myeloma that the immunophenotype of plasmoma and hereditary property seem.

5.5.2.19 light chain disease/light chain deposition diseases (LCDD)

LCDD is a kind of synthetic excessively plasmocyte dyscrasia disorder that causes by sedimentary light chain immunoglobulin (being often referred to the kappa light chain) in the tissue.The patient shows organ dysfunction, weakness, fatigue usually and loses weight.Can detect monoclonal immunoglobulin nearly in 80% the LCDD case.Available immunofluorescence technique detects mono-clonal kappa light chain, and it is subjected to being provided by light chain the restriction of excessive background dyeing tendency, and therefore, ultrastructure immuno-gold labeling thing may be necessary.(referring to: C.Wilson, The Plasma Cell Dyer asias, pp.113-144, InW.Finn and L.Peterson, eds.Hematopathology in Oncology, KluwerAcademic Publishers, Norwell, MA (2004)).

5.5.2.20 plasma cell leukemia (PCL)

PCL, a kind of plasmocyte dyscrasia is a kind of rare aggressive variant of multiple spinal cord knurl.The standard of plasma cell leukemia is that the absolute plasmocyte number of peripheral blood is greater than 2 * 10 ⁹/ L or plasmocyte are more than 20% of white blood cell.According to the restriction of tenuigenin light chain, measure the CD 138 that occurs with flow cytometry ⁺Number can pick out PCL according to plasmacytic feature from lymphoma.The feature of PCL cell also is to lack the expression of surperficial light chain and CD19, and does not express or faint expression CD45.About 50% PCL case is expressed CD20, and about 50% lacks the expression of CD56.Observed heredity is not normal with observed identical in multiple myeloma patients in PCL patient, but the frequency of finding in PCL is higher.(referring to: C.Wilson, ThePlasma Cell Dycrasias, pp.113-144, In W.Finn and L.Peterson, eds.Hematopathology in Oncology, Kluwer Academic Publishers, Norwell, MA, (2004)).

Plasma cell leukemia has two kinds of forms: if the initial stage diagnosis is based on the leukemia stage of myelomatosis, will shows the initial stage type, otherwise just belong to two types.Initial stage plasma cell leukemia and youth, hepatosplenomegaly, lymphadenopathy are relevant, compare the dissolving bone pathology that has still less with two types, but worse prognosis is arranged.Plasma cell leukemia peripheral blood of patients liquid has the plasmocyte greater than 20%, and described plasmocyte is 2000/ml or above absolute number.

5.5.2.21 the MG (MGUS) that feature is not clear

MGUS be a kind of be the relative common disease of feature with electrophoresis same sex immunoglobulin (Ig) or optimum M-component.The appearance of this illness seems to be increased with the age.The individuality that great majority carry the M-component can not develop into the malignant plasma cell dyscrasia, for example multiple spinal cord knurl.But some have the relevant malignant tumour illness of individuality of this disease.When symptom took place, the patient can have liver or splenomegaly and pleuroneuropathy.(referring to: J.Foerster, Plasma Cell Dycrasias:GeneralConsiderations, pp.2612-2630, In Wintrobe ' s Clinical Hematology, TenthEdition, people such as G.Lee, eds.Williams﹠amp; Wilkins, Baltimore, MD (1999)).

Can MGUS and multiple myeloma be distinguished by the increase of round-robin mono-clonal plasmocyte quantity in the peripheral blood.The serum characteristic of M-component is identical with other plasmocyte dyscrasia illness, and still, the total concn of M-component is usually less than 30g/L.Paraprotein is IgG normally; But multiple paraprotein may show as and comprise IgG, IgA, IgM.That finds in the general and normal serum of the relative populations of each individual immunoglobulin class is proportional.Proteinemia or proteinuria are rarely found.Blood and sequential determination, the continuous monitoring (comprising protein electrophorese) clinical and the laboratory feature of M protein level in the urine are the reliable methods of distinguishing MGUS from the early plasmocyte dyscrasia.(In Wintrobe ' s Clinical Hematology, Tenth Edition, people such as G.Lee, eds.Williams﹠amp; Wilkins, Baltimore, MD (1999)).

5.5.2.22 sophisticated B cell malignancies

The contriver has shown that the anti-CD19 composition of invention can consume sophisticated B cell.Therefore, as another aspect, can implement the present invention and treat the mature B cell malignant tumour, include but are not limited to follicular lymphoma, mantle-cell lymphoma, burkitt's lymphoma, multiple myeloma, diffustivity large B cell lymphoid tumor (DLBCL), it comprises secondary nodules B cell sample (GCB) DLBCL, activating B cell sample (ABC) DLBCL and 3 type DLBCL, hodgkin's lymphomas, it comprises typical and the trifle lymphocyte is main type, lymph-plasma cell lymphoma (LPL), marginal zone lymphoma, it comprises stomach mucous membrane in conjunction with Lymphoid tissue (MALT) lymphoma and lymphocytic leukemia (CLL), and it comprises the CLL of immunoglobulin (Ig) sudden change and the CLL that immunoglobulin (Ig) does not suddenly change.

5.5.2.23 pre B cell malignant tumour

Further, in B cell development, the CD19 ratio, for example CD20 more early expresses, so CD19 is particularly suitable for treating pre B cell and immature B cells malignant tumour, for example: in marrow.Typical pre B cell and immature B cells malignant tumour include but are not limited to lymphoma mantle cell, pre B cell acute lymphoblastic leukemia, precursor B cell lymphoblastic lymphoma and other is expressed as the malignant tumour of feature with CD19.

5.5.3 in sample or patient, measure CD19 density

When not required, the mensuration of CD19 density be can be used for further characterizing patient's diagnosis.Measure with the method for the density of cell bonded antibody known to those skilled in the art (referring to, for example: people such as Sato, J.Immunology 165:6635-6643 (2000); It discloses the method for the cell surface density of measuring special T cell differentiation antigen).Other standard method comprises that Scatchard analyzes.For example: separable antibody or fragment, carry out radio-labeling, determine that the ratio of radiolabeled antibody is lived.Then antibody is contacted with the target cell of expressing CD19.Can measure the radioactivity relevant, and, determine amount with cell bonded antibody or antibody fragment according to than work with cell.

Optionally, can use fluorescent activation cell ordering (FACS) to analyze.Usually, antibody or antibody fragment combine with the target cell of expression CD19.Add second kind of reagent with antibodies then, for example: fluorescently-labeled AIA.Can measure fluorescent color then, and density definite with it and cell bonded antibody or antibody fragment.

As another appropriate means, direct traget antibody of available detectable mark or antibody fragment, fluorophor for example, and combine with target cell.Determine mark and proteic ratio, it compared with criteria beads that described criteria beads has the mark with its bonded dose known amounts.Comparing with known standard, can be used for calculating total amount with cell bonded antibody with cell bonded mark quantity.

In a further aspect, the invention provides a kind of be used for test sample or the existence of individuality CD19 and/or the method for density in external or body.This also can be used for the effect of monitoring of diseases and treatment, and the dosage of measuring and regulate administration antibody.Can adopt imaging technique in the method in vivo, for example PET (positron emission tomography) or SPECT (single photon emission computed tomography photograph).Optionally, can be by covalently bound sequestrant indium mark anti-CD 19 antibodies.The available standards γ width of cloth is penetrated to the antibody imaging that produces, method therefor with use ZEVALIN ^TM(the anti-CD20mAb of indium mark) (Biogen Idee) gives the imaging of CD20 antigen identical.

In one embodiment, can with testing sample, optionally follow control sample, contact, realize method in the body with people's of the present invention anti-CD 19 antibodies by making under the condition that forms mixture between antibody of the present invention and people's the CD19 antigen.Detect the formation (for example: use facs analysis or Western hybridization) of mixture then.When the control sample of specimen is followed in use, in two kinds of samples, all can detect mixture, and any in the formation of mixture, the existence that all shows people's CD19 in the specimen in the statistically evident difference of sample room.

In other embodiments, can be with average fluorescent strength measuring as CD19 density.In such embodiments, the B cell is removed in patient's body, and dyeed, and measure fluorescence intensity with flow cytometry with the CD19 antibody of fluorescent mark substance markers.The average intensity of available each B cell is measured and the expression fluorescence intensity.Use such method, can compare the patient and use before and after the method and composition of the present invention treatment, or the average fluorescent strength of the representative CD19 density of hCD19 normal level between the patient and on the B cell.

After measured on the B cell CD19 express among the patient of density, the density of CD19 may influence determining of the dosage of anti-CD 19 antibodies of used the compositions and methods of the invention and/or treatment plan and/or adjust.For example: when CD19 density is high, may use the ADCC of inefficient anti-CD 19 antibodies Mediated Human.In certain embodiments, when the patient with the compositions and methods of the invention treatment has lower CD19 density, can use the anti-CD 19 antibodies of the compositions and methods of the invention of higher dosage.In other embodiments, when the patient with the compositions and methods of the invention treatment has lower CD19 density, can use anti-CD 19 antibodies than the compositions and methods of the invention of low dosage.In certain embodiments, when the patient with the compositions and methods of the invention treatment has higher CD19 density, can use anti-CD 19 antibodies than the compositions and methods of the invention of low dosage.In certain embodiments, can compare intravital CD19 density of patient and CD20 density, CD19 density can be compared with human or specific trouble patient group's average CD19 density, maybe can CD19 level CD19 density is preceding with treatment or B cell disease or disorderly premorbid patient compare.In certain embodiments, the patient who treats with the compositions and methods of the invention has the B cell malignancies, and wherein CD19 is present on the surface of B cell.

5.6 the treatment plan of immunotherapy

The anti-CD 19 antibodies composition that uses in methods of treatment/scheme as " the anti-CD19 immunotherapy " that refers to herein, can be naked antibody, immune conjugate and/or fusion rotein.Composition of the present invention can be used as single agenttherapy or unites use with other therapeutical agent or treatment plan.Can be before one or more therapeutical agent administrations, simultaneously or afterwards, carry out anti-CD 19 antibodies or immune conjugate administration.Can be used for the therapeutical agent with the treatment plan of composition of the present invention associating, comprise any inhibition or prevent cell function and/or cause the material of cytoclasis.Example includes but are not limited to, radio isotope, chemotherapeutics and toxin, for example: the enzymatic activation toxin of bacterium, fungi, plant or animal-origin, or its fragment.

Available genetically modified animal model, for example hereinafter the described mouse model of 6.2 joints is tested treatment plan described herein, or the effectiveness of any treatment plan of wanting, and described mouse model is expressed interpolation or is replaced the antigenic people CD19 of natural CD19 antigen.Therefore, can in animal model, test the anti-CD 19 antibodies treatment plan, come before to human administration, to determine to render a service.

Can adopt anti-CD 19 antibodies of the present invention, composition and method treatment B cell disease, comprise the B cell malignancies.Term " B cell malignancies " comprises the malignant tumour of any B of deriving from cell line cell.The typical B cell malignancies comprises, but be not limited only to: B cell subsets non-Hodgkin lymphomas (NHL), it comprises rudimentary/folliculus NHL, small lymphocyte (SL) NHL, middle rank/folliculus NHL, middle rank diffusion NHL, senior immunoblast NHL, senior lymphocytoblast NHL, senior small non-cleaved cell NHL; Mantle-cell lymphoma and huge sick NHL; Burkitt's lymphoma; Multiple myeloma; Preceding B acute lymphoblastic leukemia and other malignant tumour derived from early stage B cell precursor cell; Common acute Lymphocytic leukemia (ALL); Lymphocytic leukemia (CLL), it comprises immunoglobulin (Ig) sudden change CLL and the immunoglobulin (Ig) CLL that do not suddenly change; Hairy cell leukemia; Non-acute lymphoblastic leukemia; The Fahrenheit macroglobulinemia; The large B cell lymphoid tumor (DLBCL) of diffusion, it comprises secondary nodules B cell sample (GCB) DLBCL, activating B cell sample (ABC) DLBCL and 3 type DLBCL; The prolymphocyte leukemia; Light chain disease; Plasmoma; The osteosclerosis myelomatosis; Plasma cell leukemia; The MG (MGUS) that feature is not clear; Smoulder multiple myeloma (SMM); The multiple myeloma of making slow progress (IMM); Hodgkin's lymphomas, it comprises typical and the trifle prolymphocyte is main type; Lymph-plasma cell lymphoma (LPL); And marginal zone lymphoma, it comprises Lymphoid tissue (MALT) lymphoma that stomach mucous membrane is relevant.

The inventor has showed that antibody of the present invention and composition can consume mature B cell.Therefore, on the other hand, the present invention can be used for treating mature B cell malignant tumour (that is: at cell surface expression Ig), described mature B cell malignant tumour includes but are not limited to, follicular lymphoma, mantle-cell lymphoma, burkitt's lymphoma, multiple myeloma, the large B cell lymphoid tumor (DLBCL) of diffusion, it comprises secondary nodules B cell sample (GCB) DLBCL, activating B cell sample (ABC) DLBCL, and 3 type DLBCL, hodgkin's lymphomas, it comprises typical and the trifle prolymphocyte is main type, lymph-plasma cell lymphoma (LPL), marginal zone lymphoma, it comprises stomach mucous membrane-associating Lymphoid tissue (MALT) lymphoma, and lymphocytic leukemia (CLL), it comprises immunoglobulin (Ig) sudden change CLL and the immunoglobulin (Ig) CLL that do not suddenly change.

Further, CD19 is expression ratio in B cell development, for example CD20 early, so CD19 is particularly suited for treating pre B cell and immature B cells malignant tumour (that is: not at cell surface expression Ig), for example: in marrow.Pre B cell of enumerating and immature B cells malignant tumour include but are not limited to acute lymphoblastic leukemia.

In other specific embodiment, the present invention can be used for treating the outer tumour of tubercle.

5.6.1 anti-CD19 immunotherapy

" anti-CD19 immunotherapy " comprises according to any treatment plan described herein, the administration of any anti-CD 19 antibodies of the present invention according to the present invention.Anti-CD 19 antibodies can be used as naked antibody or immune conjugate or fusion rotein administration.

Anti-CD19 immunotherapy comprises the administration as the anti-CD 19 antibodies of the single agenttherapy of treatment B cell malignancies.Anti-CD19 immunotherapy comprises the method for the early stage disease that treatment is caused by the B cell malignancies.Anti-CD19 immunotherapy comprises the method for treatment B cell malignancies, wherein anti-CD 19 antibodies mediation ADCC.Anti-CD19 immunotherapy comprises the method for treatment B cell malignancies, wherein accept anyly to carry out the anti-CD 19 antibodies administration before to the treatment of malignant tumour the patient, though therapy described herein be chemotherapy, based on radiochemical therapy or surgical treatment.

In preferred embodiments, can treat the patient who suffers from the B cell malignancies, the people's or the preferred Mediated Human ADCC's of humanized antibody antibody described herein by the people's or humanized antibody administration.If early stage disease or single agenttherapy can use any anti-CD 19 antibodies to human patients, preferably mediate the antibody (comprise mouse with chimeric antibody) of ADCC; But, the preferred people and humanized antibody.

Preferred IgG1 or IgG3 people's isotype antibody are used for the treatment of.But, can use IgG2 or IgG4 people's isotype, as long as the ADCC of its Mediated Human.Can in external or body, mediate the ability that target cell is dissolved by the effector cell by measuring the antibody of discussing, measure the function of this effector.

The dosage of used antibody should enough consume circulation B cell.Can come monitor therapy process in the patient by the analyzing blood sample.Other of available improvement clinically indicates to come monitor therapy.

Be known in the art the method that can be used for the mensuration B cell consumption of the compositions and methods of the invention associatings, it includes but are not limited to following embodiment.In one embodiment, can pass through flow cytometry, use a kind of reagent that except anti-CD 19 antibodies, combines to determine the B cell concentration, the consumption of measuring circulation B cell with the B cell.In other embodiment, the available standards serum analysis is come the antibody horizontal in the monitoring of blood.In such embodiments, by defining the quantity of the known antibody that produces by the B cell, directly measure the B cell consumption.Monitor the level of antibody then, determine the consumption and/or the functional consumption of B cell.In another embodiment, available immunochemistry dyeing affirmation B cell is measured the B cell consumption.In such embodiments, the B cell of taking from patient tissue can be placed on the microslide, carry out mark, and inspection exists or do not exist.In relevant embodiment,, measure the difference that the B cell exists to comparing with the B cell that extracts afterwards before the treatment.

Can measure tumor load, and with itself and the compositions and methods of the invention combined utilization.The method of known mensuration tumor load in this area includes but are not limited to following embodiment.In certain embodiments, available PET sweep measuring metabolic activity, and the highly active zone of affirmation indication tumour.Also available CT scan and MRI come the detected representation tumour to exist and big or small soft tissue.In other embodiment, available bone scan is measured tumor size and position.In other embodiments, available Doppler technology (for example: ultrasonic) detects the blood flow of turnover tumour, measures tumor load.In such embodiments, the available blood flow that changes in time changes or estimates tumor load with the departing from of normal blood flow that the patient suitably organizes.Can before or after using methods of treatment of the present invention, use the method for this mensuration tumor load.

In the preferred embodiment of method of the present invention, consume the B cell and/or reduce tumor load, and keep the ADCC function.

In embodiments of the invention, when anti-CD 19 antibodies was treated administration as single agent, the present invention had considered the use of different treatment plans.

According to some aspect of the present invention, be used for the anti-CD 19 antibodies of the compositions and methods of the invention, be naked antibody.In relevant embodiment, the dosage of used naked anti-CD 19 antibodies is at least about 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1,1.5,2,2.5,3,3.5,4,4.5,5,5.5,6,6.5,7,7.5,8,8.5,9,9.5,10,10.5,11,11.5,12,12.5,13,13.5,14,14.5,15,15.5,16,16.5,17,17.5,18,18.5,19,19.5,20, the 20.5mg of every kg weight in patients.In certain embodiments, the dosage of used naked anti-CD 19 antibodies is at least every kg weight in patients about 1 to 10,5 to 15,10 to 20 or 15 to 25mg.In certain embodiments, the dosage of used naked anti-CD 19 antibodies is at least every kg weight in patients about 1 to 20,3 to 15 or 5 to 10mg.In preferred embodiments, the dosage of used naked anti-CD 19 antibodies is at least every kg weight in patients about 5,6,7,8,9 or 10mg.

In certain embodiments, dosage comprises about 375mg/m ²Anti-CD 19 antibodies, continuous 4 to 8 weeks administration weekly.In certain embodiments, dosage is at least every kg weight in patients about 1,2,3,4,5,6,7,8,9,10,11,12,13,14 or 15mg, continuous 4 to 8 weeks administration weekly.

The exemplary dose of aforesaid anti-CD 19 antibodies can save described administration by 5.4.3.In one embodiment, above-mentioned dosage is single agent injection.In other embodiment, dosage is administration in for some time.In other embodiment, dosage is multiple dosing in for some time.This section period can calculate by sky, the moon or week.The multiple doses of anti-CD 19 antibodies can be to be suitable for reaching result of treatment, and balance toxic side effect and the administration at set intervals taked.For example, when using multiple doses, preferably set before, make the pitch time of patient's monocyte number recovery with the antibody repetitive therapy.The treatment plan of this dosage will be optimized therapeutic efficiency, because the monocyte number reflects patient's ADCC function.

In certain embodiments, as long as the patient responds to treatment, just human patients is carried out the administration of composition of the present invention.In other embodiment,, just human patients is carried out the administration of composition of the present invention as long as disease of patient does not develop.In relevant embodiment, human patients is carried out the administration of composition of the present invention, do not develop or not development in for some time up to disease of patient, this patient is not carried out the administration of composition of the present invention then, unless palindromia or begin development again.For example: available above-mentioned any dosage carries out the treatment of 4 to 8 weeks to the patient, during this period, and to the patient-monitoring disease progression.If disease progression stops or taking a turn for the better, then this patient is not carried out the administration of composition of the present invention, unless patient recurrence, that is: the palindromia of treatment or developed.When recurrence or when development, the treatment plan of the dosage that available and initial use is identical or use other above-mentioned dosage to treat the patient.

In certain embodiments, synthetics of the present invention can be in for some time multiple low dose (maintenance dosage) afterwards as the loading dose administration.In such embodiments, be the B cell consumption of remaining valid, can carry out timing and dose adjusting dosage.In preferred embodiments, loading dose is every kg weight in patients about 10,11,12,13,14,15,16,17 or 18mg, and maintenance dose is every kg weight in patients about 5 to 10mg.In preferred embodiments, carried out the maintenance dose administration every 7,10,14 or 21 days.Maintenance dose can irregularly continue, up to show toxicity, up to platelet count reduce, up to disease no longer develop, up to the patient medicine is produced immune response or up to disease progression to latter stage.In other embodiment, human patients is carried out the administration of composition of the present invention, up to disease progression to latter stage.

In embodiments of the invention, when monitoring patient's circulating monocytic cell level became treatment plan a part of, the dosage of anti-CD 19 antibodies can be spaced apart, so that the monocyte amount is recovered.For example: can carry out administration to composition of the present invention every 8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or 30 days.

In embodiments of the invention, when anti-CD 19 antibodies and toxin conjugation or Combined Preparation, those skilled in the art can understand the dosage that can regulate anti-CD 19 antibodies according to toxin dose, and this toxin dose depends on the particular types of used toxin.Usually, when using toxin, the dosage of anti-CD 19 antibodies is less than dosage used when using naked anti-CD 19 antibodies.Available technology known in the art is determined the suitable dosage of particular toxin.For example: can adopt dosage range research to measure to determine when anti-CD 19 antibodies and toxin Combined Preparation or conjugation the limit tolerance of anti-CD 19 antibodies.

In embodiments of the invention, when anti-CD 19 antibodies and radiotherapy reagent conjugation or Combined Preparation, the dosage of anti-CD 19 antibodies will change according to used radiotherapy.In certain preferred aspects, use two-stage process.The first, human patients is comprised the administration of the composition of naked anti-CD 19 antibodies, and after about 6,7,8,9 or 10 days, carry out a small amount of radiotherapy.The second, in case measured tolerance, distribution and the clearance rate of low dose therapy, after giving the radiotherapy dose of certain therapeutic dose, give naked anti-CD 19 antibodies to the patient.Such treatment plan is similar to those approved use ZEVALIN ^TM(the anti-CD20mAb of indium mark) (Biogen Idee) or BEXXAR ^TMThe treatment that (GSK, Coulter Pharmaceutical) carries out the non-Hodgkin lymphomas.

5.6.2 with combining of chemotherapeutics

Can will resist CD19 immunotherapy (using naked antibody, immune conjugate or fusion rotein) and other therapy to unite use, described other therapy includes but are not limited to, chemotherapy, radioimmunoassay therapy (RIT), chemotherapy and external beam radiotherapy (blended form therapy, CMT) or blended form radioimmunoassay therapy (CMRIT), separately or in conjunction with etc.In certain preferred aspects, can be with anti-CD 19 antibodies therapy of the present invention and CHOP (endoxan-Hydroxydoxorubicin-vincristine(VCR) (vincristine)-Prednisolone Acetate) Combined Preparation, the modal chemotherapy regimen of treatment non-Hodgkin lymphomas.As used herein, term " Combined Preparation " refer to anti-CD19 immunotherapy can be before another therapy of using, simultaneously or administration afterwards.

In certain embodiments, anti-CD19 immunotherapy and cytotoxic radionuclide or radiotherapeutic isotropic substance are united use.For example: alpha ray isotope, for example: ²²⁵Ac, ²²⁴Ac, ²¹¹At, ²¹²Bi, ²¹³Bi, ²¹²Pb, ²²⁴Ra or ²²³Ra.Optionally, cytotoxic radionuclide may be a beta-emitting isotope, for example: ¹⁸⁶Re, ¹⁸⁸Re, ⁹⁰Y, ¹³¹I, ⁶⁷Cu, ¹⁷⁷Lu, ¹⁵³Sm, ¹⁶⁶Ho or ⁶⁴Cu.And cytotoxic radionuclide can send Auger and low-energy electron, comprising: isotropic substance ¹²⁵I, ¹²³I or ⁷⁷Br.In other embodiment, isotropic substance can be ¹⁹⁸Au, ³²P etc.In certain embodiments, to the amount of the radionuclide of object administration at about 0.001mCi/kg between about 10mCi/kg.

In some preferred embodiments, to the amount of the radionuclide of object administration at about 0.1mCi/kg between about 1.0mCi/kg.In other embodiment preferred, to the amount of the radionuclide of object administration at about 0.005mCi/kg between the 0.1mCi/kg.

In certain embodiments, anti-CD19 immunotherapy and chemical toxicant or chemotherapeutics are united use.Preferably chemical toxicant of selecting from the group of being made up of the enediyne class or chemotherapeutics, described enediyne class be for example: calicheamycin and Ai Sibo mycin (esperamicin); Duocarmycin, methotrexate, Zorubicin, Melphalan, Chlorambucil, ARA-C, desacetyl vinblastine amide, ametycin, suitable-platinum, etoposide, bleomycin and 5 FU 5 fluorouracil.

Can unite the suitable chemical toxicant of use or the member that chemotherapeutics comprises enediyne class family molecule with anti-CD19 immunotherapy, for example: calicheamycin and Ai Sibo mycin (esperamicin).Also can from the group of forming by duocarmycin, obtain chemical toxicant (referring to, for example: U.S. Patent number 5,703,080 and U.S. Patent number 4,923,990), methotrexate, Zorubicin, Melphalan, Chlorambucil, ARA-C, desacetyl vinblastine amide, ametycin, suitable-platinum, etoposide, bleomycin and 5 FU 5 fluorouracil.The example of chemotherapeutics also comprises Zorubicin, Zorubicin, 5 FU 5 fluorouracil, cytosine arabinoside (" cytosine arabinoside "), endoxan, thiotepa, taxotere (Japanese yew terpene), busulfan, endoxan, taxol, methotrexate, Platinol, Melphalan, vincaleucoblastine, bleomycin, etoposide, ifosfamide, ametycin, mitoxantrone, Vincreistine, Vinorelbine, carboplatin, Vumon, Rubomycin C, carminomycin, aminopterin, gengshengmeisu, mitomycin, Ai Sibo mycin (esperamicin) (referring to: U.S. Patent number 4,675,187), Melphalan and other relevant nitrogen mustards.

In other embodiment, for example: " CVB " (1.5g/m ²Endoxan, 200-400mg/m ²Etoposide and 150-200mg/m ²Carmustine) can be used for combination therapy of the present invention.CVB is a kind of treatment plan that is used for treating the non-Hodgkin lymphomas.People such as Patti, Eur.J.Haematol.51:18 (1993).The chemotherapy regimen of known other the suitable associating of those skilled in the art.Referring to, for example: people such as Freedman, " Non-Hodgkin ' s Lymphomas " inCANCER MEDICINE, VOLUME 2,3rd Edition, people such as Holland (eds.), pp.2028-2068 (Lea﹠amp; Febiger 1993).As illustration, the first-generation chemotherapy regimen of treatment moderate non-Hodgkin lymphomas comprises C-MOPP (endoxan, vincristine(VCR), procarbazine and prednisone) and CHOP (endoxan, Zorubicin, vincristine(VCR) and prednisone).Effectively s-generation chemotherapy regimen is m-BACOD (methotrexate, bleomycin, AC, vincristine(VCR), dexamethasone and a formyl tetrahydrofolic acid), and suitable third generation treatment plan is MACOP-B (methotrexate, AC, vincristine(VCR), prednisone, bleomycin and a formyl tetrahydrofolic acid).Other active drug comprises phenyl butyrate and brostatin-1.In preferred multiple mode therapy, chemotherapeutics and cytokine and according to antibody of the present invention, immune conjugate or fusion rotein co-administered.Cytokine, chemotherapeutics and antibody, immune conjugate or fusion rotein can be by any order or co-administereds.

Preferred other toxin that uses comprises poisonous Sugar receptors, plant poison in the compositions and methods of the invention, for example: ricin, toxalbumin, modeccin, Toxins, botulin and diphtheria toxin.Certainly, also the built up section of various toxin can be arrived antibody molecule, thereby regulate variable cytotoxicity.The example that is applicable to the toxin of conjoint therapy of the present invention is ricin, toxalbumin, rnase, DNase I, staphyloentero-toxin-A, pokeweed antiviral protein, spends more white tree inhibitor (gelonin), diphtheria toxin, Rhodopseudomonas extracellular toxin and Rhodopseudomonas intracellular toxin.Referring to, for example: people such as Pastan, people such as Cell 47:641 (1986) and Goldenberg, Cancer Journal for Clinicians 44:43 (1994).Available enzymatic activation toxin and fragment thereof comprise diphtheria A chain, the not combination activation fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-Zhou Qujunsu, tung oil tree albumen, alizarin albumen, dyers' grapes albumen (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, spend more white tree inhibitor (gelonin), mitogellin, restrictocin, phenomycin, enomycin and tricothecenes.Referring to, for example, on October 28th, 1993 disclosed WO93/21232.

At REMINGTON ' S PHARMACEUTICAL SCIENCES, 19th Ed. (MackPublishing Co.1995) and GOODMAN AND GILMAN ' S THEPHARMACOLOGICAL BASIS OF THERAPEUTICS have described suitable toxin and chemotherapeutics among the 7th Ed. (MacMillanPublishing Co.1985).Those skilled in the art known other suitable toxin and/or chemotherapeutics.

Anti-CD19 immunotherapy of the present invention also can combine with the prodrug activation enzyme, described prodrug activation enzyme with prodrug (for example: the peptidyl chemotherapeutics, referring to: WO81/01145) be converted into activated carcinostatic agent.Referring to, for example: WO 88/07378 and U.S. Patent number 4,975,278.The enzyme component of such composition comprises anyly can act on prodrug by this way so that it changes any enzyme of more activated cytotoxin form into.Term " prodrug " refers to the precursor or the derivative form of pharmaceutically active substance as used in this application, and it is compared with parent drug for tumour cell has still less cytotoxicity, and can the enzymatic activation or change more activated parent drug form into.Referring to, for example, Wilman, " Prodrugs in CancerChemotherapy ", Biochemical Society Transactions, 14, pp.375-382, people such as 615thMeeting Belfast (1986) and Stella, " Prodrugs:A Chemical4pproachto Targeted Drug Delivery ", Directed Drug Delivery, people such as Borchardt (ed.) 5, pp.247-267, Humana Press (1985).Can be used for comprising with the prodrug of anti-CD 19 antibodies combination of the present invention, but be not limited to, comprise phosphatic prodrug, the prodrug that comprises thiophosphate, the prodrug that comprises vitriol, comprise the propeptide medicine, the prodrug that D-is amino acid modified, glycosylated prodrug, the prodrug that comprises α-lactan, selectivity replaces the prodrug or the selectivity that comprise phenoxyacetamide and replaces the prodrug that comprises phenylacetamide, 5-flurocytosine and other can change the 5-floxuridine prodrug of the free agent of more activated cytotoxin into.The example that can be derivatized to the cytotoxic drug of prodrug form used among the present invention includes, but not limited to aforesaid those chemotherapeutics.

In some concrete embodiment, the administration of the compositions and methods of the invention can make toxic agent treatment delay, and can help avoid unnecessary side effect and with the danger of chemotherapy complications associated with arterial system, and postpone development for the chemotherapy resistance.In some concrete embodiment, by giving the compositions and methods of the invention to the patient, the resistance of having postponed the toxic agent treatment and/or toxic agent being treated is deferred to about 6 months, 1,2,3,4,5,6,7,8,9 or 10 year with it.

5.6.3 the combination of therapeutic antibodies

Anti-CD19 immunotherapy described herein can with other antibodies administration, described other antibody includes but not limited to, anti-CD20mAb, anti-CD52mAb, anti-CD22 antibody (for example, as at U.S. Patent number 5,484,892, US application serial No. 10/371,797 U.S. Patent Publication No. 2004/0001828, US application serial No. 10/372,481 U.S. Patent Publication No. 2003/0202975 and U.S. Provisional Application sequence number 60/420, described in 472, wherein the full content of each reference is incorporated into herein as a reference, is used for the instruction of CD22 antigen and anti-CD22 antibody), and anti-CD20 antibodies, for example: RITUXAN ^TM(C2B8; RITUXIMAB ^TMBiogen Idee).Can be used for and antibody of the present invention combination, or other example that is used for the therapeutic antibodies of composition of the present invention includes, but not limited to HERCEPTIN ^TM(Trastuzumab; Genentech), MYLOTARG ^TM(Gemtuzumab ozogamicin; Wyeth Pharmaceuticals), CAMPATH ^TM(Alemtuzumab; Berlex), ZEVALIN ^TM(Ipritumomabtiuxetan; Biogen Idee), BEXXAR ^TM(Tositumomab; GlaxoSmithKlineCorixa), ERBITUX ^TM(Cetuximab; Imclone) and AVASTIN ^TM(Bevacizumab; Genentech).

In certain embodiments, anti-CD19 and anti-CD20 and/or anti-CD22mAb are optionally in identical pharmaceutical composition, with any proper proportion administration.Explanation for example, the ratio of anti-CD19 and anti-CD20 antibodies can be about 1000: 1,500: 1,250: 1,100: 1,90: 1,80: 1,70: 1,60: 1,50: 1,40: 1,30: 1.20: 1,19: 1,18: 1,17: 1,16: 1,15: 1,14: 1,13: 1,12: 1,11: 1,10: 1,9: 1,8: 1,7: 1,6: 1,5: 1,4: 1,3: 1,2: 1,1: 1,1: 2,1: 3,1: 4,1: 5,1: 6,1: 7,1: 8,1: 9,1: 10,1: 11,1: 12,1: 13,1: 14,1: 15,1: 16,1: 17,1: 18,1: 19,1: 20,1: 30,1: 40,1: 50,1: 60,1: 70,1: 80,1: 90.1: 100,1: 250,1: 500 or 1: 1000 or above ratio.Similarly, the ratio of anti-CD19 and anti-CD22 antibody can be about 1000: 1,500: 1,250: 1,100: 1,90: 1,80: 1,70: 1,60; 1,50: 1,40: 1,30: 1.20: 1,19: 1,18: 1,17: 1,16: 1,15: 1,14: 1,13: 1,12: 1,11: 1,10: 1,9: 1,8: 1,7: 1,6: 1,5: 1,4: 1,3: 1,2: 1,1: 1,1: 2,1: 3,1: 4,1: 5,1: 6,1: 7,1: 8,1: 9,1: 10,1: 11,1: 12,1: 13,1: 14,1: 15,1: 16,1: 17,1: 18,1: 19,1: 20,1: 30,1: 40,1: 50,1: 60,1: 70,1: 80,1: 90.1: 100,1: 250,1: 500 or 1: 1000 or above ratio.

5.6.4 strengthen the composition of monocyte or macrophage function

In some embodiment of the inventive method, the composition that will increase monocyte or scavenger cell number or function (for example: about at least 25%, 50%, 75%, 85%, 90%, 95% or more than) is used for combining with anti-CD19 immunotherapy.At such composition known in the art, it comprises, is not limited to, and for example interleukin is (for example: IL-12) and Interferon, rabbit (for example: α or IFN-) for cytokine.

Can strengthen the composition of monocyte or macrophage function or enhancement as preparation in antibody, immune conjugate or the Fab at identical pharmaceutical composition.When the difference administration, the administration (each other within several hours time) simultaneously of antibody/fragment and composition, can the treatment identical process in administration, (that is: the patient at first accepts a series of antibody/fragment treatment in perhaps administration in proper order, accept to strengthen a series of combination treatments of treatment monocytes/macrophages function then, or vice-versa).In such embodiments, before with other treatment plan of the present invention and/or combination treatment, simultaneously or afterwards, give the testee with the composition that strengthens monocyte or macrophage function.In one embodiment, the testee has blood leucocyte, monocyte, neutrophil, lymphocyte and/or the basophilic leukocyte number in the human body normal range.The normal range of human blood white corpuscle (sum) is about 3.5-about 10.5 (10 ⁹/ L).The normal range of human blood neutrophil is about 1.7-about 7.0 (10 ⁹/ L), monocyte is about 0.3-about 0.9 (10 ⁹/ L), lymphocyte is about 0.9-about 2.9 (10 ⁹/ L), basophilic leukocyte is about 0-about 0.3 (10 ⁹/ L), eosinophilic granulocyte is about 0.05-about 0.5 (10 ⁹/ L).In other embodiment, the testee has the blood leucocyte number that is less than the human body normal range, and for example about at least 0.01,0.05,0.1,0.2,0.3,0.4,0.5,0.6,0.7 or 0.8 (10 ⁹/ L) white corpuscle.

This embodiment of the present invention can be implemented with antibody of the present invention, immune conjugate or antibody fragment or other antibody known in the art, is particularly suitable for resisting object that CD19, anti-CD20 and/or anti-CD22 Antybody therapy (for example: use for example treatment of C2B8 of existing antibody) have resistance, current or before with the object of the object of the object of chemotherapeutic treatment, the recurrence of B cell disorder, immunological competence infringement or defective object on scavenger cell or monocyte function in addition.Treatment there be patient popular of resistance or B cell disorder recurrence, at least can be partly owing to the defective on scavenger cell or monocyte function.Therefore, the invention provides and strengthen ADCC and/or scavenger cell and/or monocyte function and be used for the method that combines with the method that gives anti-CD 19 antibodies and Fab.

5.6.5 with the immunomodulatory combination of agents

Anti-CD19 immunotherapy of the present invention also can be used for and the immunomodulatory combination of agents.In the method, preferably use chimeric antibody; Most preferably the end user's or humanized anti-CD 19 antibodies.The term " immunomodulatory reagent " that is used for combination therapy herein refers to bear inhibition, shelter or strengthen the material of host immune system.It comprises that suppressing cytokine produces, reduces or suppress autoantigen and express or shelter the antigenic material of MHC.The example of such reagent comprises 2-amino-6-aryl-5-substituted pyrimidines (referring to U.S. Patent number 4,665,077), imuran (or endoxan, if having reversed reaction for imuran); Bromocriptine; Glutaraldehyde (as U.S. Patent number 4,120,649 is described, and it shelters MHC antigen); Anti-specific antibody, for example: prednisone, methyl meticortelone and dexamethasone; Cytokine or cytokine receptor antagonist body, it comprise anti-interferon-γ ,-β or-Alpha antibodies; Anti-tumor necrosis factor-Alpha antibodies; Anti-tumor necrosis factor-β antibody; The antibody of anti-interference leukin-2 antibody and anti-IL-2 acceptor; Anti-L3T4 antibody; The xenogeneic antilymphocyte globulin (ALG); Full T antibody, preferred anti-CD3 or anti-CD4/CD4a antibody; Soluble peptide, it comprises LFA-3 binding domains (disclosed WO 90/08187 on July 26 nineteen ninety); Streptokinase; TGF-β; Streptodornase; RNA or DNA from the host; FK506; RS-61443; The deoxidation spergualin; Rapamycin; TXi Baoshouti (U.S. Patent number 5,114,721); TXi Baoshouti fragment (people such as Offner, Science251:430-432 (1991); WO 90/11294 and WO 91/01133); And the antibody of TXi Baoshouti (EP340,109), for example T10B9.The example of cytokine includes, but are not limited to lymphokine, unicellular live plain and traditional polypeptide hormone.In cytokine, comprise tethelin, for example: human growth hormone, N-methionyl human growth hormone and Polisac; Rat parathyroid hormone 1-34; Thyroxine; Regular Insulin; Proinsulin; Relaxin; Relaxation precipitinogen; Glycoprotein hormones, for example: follicle stimulating hormone (FSH), thyrotropic hormone (TSH) and prolan B (LH); The liver somatomedin; Fibroblast growth factor; Prolactin; The placenta lactogen; Tumor necrosis factor-alpha; The mullerian-inhibitory substance; Mouse gonad-stimulating hormone binding peptide; Statin; Nrolone Phenylpropionate; The vascular endothelial cell growth factor (ECGF); Integrate plain; Thyroid peroxidase (TPO); Nerve growth factor, for example NGF-α; PDGF; Transforming growth factor (TGFs), for example TGF-α and TGF-α; Insulin like growth factor-1 and-II; Erythropoietin (EPO); Osteoinductive factors; Interferon, rabbit; G CFS (CSFs), for example scavenger cell-CSF (M-CSF); Granulocyte-macrophage-CgP (GM-CSP) and granulocyte-CSF (G-CSF); Interleukin (ILs), for example IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; Tumour necrosis factor, for example TNF-α or TNF-β; And other polypeptide factor, comprise LIF and kit aglucon (KL).As used herein, the term cytokine comprises from natural origin or from the albumen of reconstitution cell culture and the biologically activated equivalent of native sequences cytokine.In certain embodiments, this method comprises further and gives one or more immunomodulatory reagent to object that preferred cell is lived plain.Preferring cytokine is to elect from the group of being made up of il-1 (IL-1), IL-2, IL-3, IL-12, IL-15, IL-18, G-CSF, GM-CSF, thrombopoietin and IFN-.

These immunoregulatory reagent are with the anti-CD 19 antibodies while of the present invention or at the time administration that separates, and dosage more identical than the use of this area proposition or still less.Preferred immunomodulatory reagent relies on many factors, the kind that comprises the illness of treatment, and patient's medical history, but the reagent of generally generally speaking preferably from cyclosporin A, glucocorticosteroid (most preferably prednisone or methyl meticortelone), OKT-3 monoclonal antibody, imuran, bromocryptine, xenogeneic antilymphocyte globulin (ALG) or its mixture, electing.

5.6.6 combination with other therapeutical agent

The reagent that acts on the tumour neovasculature can also be used for combining with anti-CD19 immunotherapy, and comprise the tubulin tackiness agent, health nurse Greensboro sthene (combrestatin) A4 (people such as Griggs for example, Lancet Oncol.2:82 (2001)), (summary is at Rosen for Anji Ao Sitayan (angiostatin) and En Duositayan (endostatin), Oncologist 5:20, in 2000, incorporate into herein as a reference).Immunomodulator is suitable for uniting use with anti-CD 19 antibodies, and described anti-CD 19 antibodies includes, but not limited to alpha-interferon, gamma-interferon and tumor necrosis factor alpha (TNF α).In certain embodiments, the therapeutical agent that is used for the use the compositions and methods of the invention of combination therapy is a peptide.

In certain embodiments, anti-CD19 immunotherapy and one or more calicheamycin molecules associating.The calicheamycin family of antibiotic can produce the double-stranded DNA breakpoint in set place of submicron/micron.The analog of spendable calicheamycin comprises, but be not limited to, γ 11, γ 21, γ 31, N-acetyl-γ 11, PSAG and 011 (people such as Hinman, people such as Cancer Research 53:3336-3342 (1993) and Lode, Cancer Research 58:2925-2928 (1998)).

Optionally, can synthesize by for example recombinant technology or peptide and prepare the fusion rotein that comprises anti-CD 19 antibodies of the present invention and cytotoxin reagent.

In another embodiment, anti-CD 19 antibodies of the present invention can with " acceptor " (for example: streptavidin) combine and be used for target-seeking before the tumour, wherein give the patient with antagonist acceptor conjugate, use finings subsequently and remove unconjugated conjugate from circulation, (for example: " aglucon " radioactivity nucleic acid) (for example: administration vitamin H) will to be attached to therapeutical agent then.

In certain embodiments, treatment plan comprises the composition of the cytotoxin effect that alleviates anti-CD 19 antibodies composition of the present invention.Such composition comprise pain killer (for example: acetaminophen), diphosphonate, antihistaminic agent (for example: Toldrin) and steroid (for example: dexamethasone, retinoid, deltoid plate, Betamethasone Valerate, hydrocortisone, cortisone, prednisone, 2-boldenone, glucocorticosteroid, mineralocorticoid, oestrogenic hormon, testosterone, progesterone).

In certain embodiments, the therapeutical agent of uniting use with anti-CD19 immunotherapy of the present invention is small molecules (for example: have the inorganic or organic compound less than about 2500 Dalton molecular weights).For example: micromolecular storehouse can be commercial from Specs and BioSpecs B.V. (Rijswijk, Holland), Chembridge Corporation (San Diego, CA), ComgenexUSA Inc. (Princeton, NJ) and Maybridge Chemicals Ltd. (Cornwall PL34OHW, Britain) locate to obtain.

In certain embodiments, anti-CD19 immunotherapy can combine administration with antibacterium reagent.The non-limitative example of antiseptic-germicide comprises and suppresses and/or reduce bacterial infection and/or reduce that bacterium is duplicated or and/or reduce cell or protein in object, polypeptide, peptide, fusion rotein, antibody, nucleic acid molecule, organic molecule, inorganic molecule and the small molecules of spread of germs to other.The specific example of antiseptic-germicide comprises, but be not limited to, antibiotic is penicillin, cynnematin, imipenum, aztreonam, vancomycin, seromycin, bacitracin, chloroacetic, erythromycin, clindamycin, tsiklomitsin, Streptomycin sulphate, tobramycin, gentamicin, amikacin, kantlex, Xin Meisu, trobicin, trimethoprim, norfloxicin, Rifampin, polymyxin, amphotericin B, nystatin, KETOKONAZOL, vazadrine, metronidazole and pentamidine for example.

In certain embodiments, anti-CD19 immunotherapy of the present invention can combine administration with antifungal agents.The specific examples of anti-mycotic agent (for example: miconazole, KETOKONAZOL (NIZORAL includes, but not limited to pyrroles's reagent

), caspofungin acetate (CANCID AS

), imidazoles, triazole (for example: fluconazole (DIFLUCAN

) and itraconazole (SPORANOX

), polyenoid (for example: nystatin, amphotericin B (FUNGIZONE ), amphotericin B class fat complexes) (" ABLC ") (ABELCET

), amphotericin B colloidal dispersion (" ABCD ") (AMPHOTEC

), liposome amphotericin b (AMBISONE

), potassiumiodide (KI), pyrimidine (for example: 5-flurocytosine (ANCOBON

) and voriconazole (VFEND).The administration of antibacterium and antifungal agents can alleviate the influence or the expansion of transmissible disease, and it may take place in the method for the invention when obviously having consumed patient's B cell.

In certain embodiments, anti-CD19 immunotherapy of the present invention can with aforesaid one or more reagent Combined Preparation, alleviate the toxic side effects that may follow present composition administration.In other embodiment, anti-CD19 immunotherapy of the present invention can be used to alleviate the reagent Combined Preparation of antibody administration, chemotherapy, toxin or drug side effect with known in the art one or more.

In certain embodiments of the invention, anti-CD19 immunotherapy of the present invention is treated multiple myeloma, composition of the present invention can be united with following treatment, or in treatment plan, comprise following treatment: high dosage chemotherapy (melphalan, melphalan/prednisone (MP), vincristine(VCR)/Zorubicin/dexamethasone (VAD), liposomal doxorubicin/vincristine(VCR), dexamethasone (DVd), endoxan, etoposide/dexamethasone/cytosine arabinoside, Platinol (EDAP), stem cell transplantation (for example: autologous stem cell transplantation or allosome stem cell transplantation, and/or little allosome (non-marrow separation) stem cell transplantation, radiotherapy, steroid (for example: corticosteroid, dexamethasone, Thalidomide/dexamethasone, prednisone, melphalan/prednisone), supportive treatment (for example: diphosphonate, somatomedin, antibiotic, the intravenously immunoglobulin (Ig), low dosage radiotherapy and/or plastic surgery intervention), THALOMID ^TM(tranquilizer, Celgene) and/or VELCADE ^TM(bortezomib, Millennium).

In embodiments of the invention, anti-CD19 immunotherapy of the present invention combines administration with another or a plurality of antibody and/or reagent, and one or more antibody of increase and/or reagent can carry out administration with respect to any order of the administration of antibody of the present invention.For example, the one or more antibody that increase for human subjects of the present invention or immune conjugate can be before the anti-CD 19 antibodies administration, simultaneously or administration afterwards.The one or more antibody that increase can be used as antibody of the present invention and are present in the identical pharmaceutical composition, or are present in the different pharmaceutical compositions.Any instruction of that provide according to the application and dosage known in the art and mode, the dosage of antibody of the present invention and mode, and the dosage of the one or more antibody that increase can be identical or different.

5.7 the application of anti-CD 19 antibodies in diagnosis B cell malignancies

The present invention also comprises anti-CD 19 antibodies and the composition thereof on the CD19 antigen that immunologic opsonin is attached to the people, and its anti-CD 19 antibodies is attached on diagnostic or the detectable reagent.In preferred specific embodiments, antibody is the people's or humanized anti-CD 19 antibodies.Such anti-CD 19 antibodies can be used as the part of clinical testing procedure, is used to monitor or predict the growth or the progress of B cell malignancies, for example: the effectiveness of determining particular treatment.Such diagnosis and detect can be attended by the anti-CD 19 antibodies that immunologic opsonin is attached on people's the CD19 antigen and to be attached on the detectable material, described detectable material includes but not limited to, various enzymes, such as but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase enzymes or acetylcholinesterase; Prothetic group, such as but not limited to, streptavidin/vitamin H and avidin/biotin; The fluorescence raw material, such as but not limited to, Umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine b extra 500, dichlorotriazine amine fluorescein, dansyl chloride or phycoerythrin; The cold light raw material, such as but not limited to, luminol; The noclilucence raw material, such as but not limited to, luciferase, fluorescent element and aequorin; Radioactive substance, such as but not limited to, iodine ( ¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon ( ¹⁴C), sulphur ( ³⁵S), tritium ( ³H), indium ( ¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In) and technetium ( ⁹⁹Tc), thallium ( ²⁰¹Ti), gallium ( ⁶⁸Ga, ⁶⁷Ga), palladium ( ¹⁰³Pd), molybdenum ( ⁹⁹Mo), xenon ( ¹³³Xe), fluorine ( ¹⁸F), 153Sm, 177Lu, 159Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y5, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge5, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr5, ³²P, ¹⁵³Gd5, ¹⁶⁹Yb5, ⁵¹Cr5, ⁵⁴Mn, ⁷⁵Se5, ¹¹³Sn5 and ¹¹⁷Tm; Utilize the emission positron of various positron radiation section layer imaging metal, labelled with radioisotope or be attached to on-radiation paramagnetic metal ion and molecule on the special radio isotope.Any detectable mark that can easily measure all can be attached on the anti-CD 19 antibodies, and is used to diagnose the B cell malignancies.Detectablely may be directly coupling or be attached on the antibody, or use methods known in the art, indirectly by intermediate (for example, connexon known in the art) coupling or be attached on the antibody.For can being attached to diagnostic metal ion on the antibody according to the present invention, referring to, for example: U.S. Patent number 4,741,900.In certain embodiments, the invention provides and comprise the diagnostic kit that is attached to the anti-CD 19 antibodies on diagnostic or the detectable reagent.

5.8 test kit

The invention provides and a kind ofly comprise one or more and be full of of the present invention being used for and prevent, treat, handle or improve the B cell malignancies, or be reinforced or strengthen the medicine assembly or the test kit of composition of one or more symptoms of B cell malignancies.

The invention provides the test kit that can be used for aforesaid method.In one embodiment, test kit is included in the of the present invention a kind of composition in one or more containers.In another embodiment, test kit be included in one or more containers of the present invention a kind of composition and in one or more containers one or more other to preventing, treat, handle or improve the B cell malignancies, or be reinforced or strengthen useful other preventive or the therapeutical agent of one or more symptoms of B cell malignancies.Preferably, test kit further comprises the guidance that is used to prevent, treat, handle or improve the B cell malignancies, and the side effect of medication and dosage information.At random the form regulation that interrelates the control manufacturing, the use that can be used as government organs or sell medicine or biological products with such container will be noted, itself since make, use or sell the people medicine effect and need obtain permitting.

6. embodiment

In following examples, transgene mouse model is used for appraiser's the immunotherapy at CD19.It all is effective that these data presentation are induced on the B cell consumption in the body of the object with the effector cell who expresses Fc γ R (preferred, Fc γ RIII or Fc γ RIV) and carry out ADCC in conjunction with the antibody of CD19 antigen and mediation ADCC.Such antibody can be used for the lasting consumption of B cell in the inductor, and can almost eliminate the B cell that all comes self-circulation, spleen and lymphatic node in certain embodiments.Unexpectedly, expression relative low density CD19 antigenic marrow B cell and precursor thereof have also been consumed.The validity of B cell consumption does not depend on which district of anti-CD 19 antibodies in conjunction with people's CD19, but is subjected to the influence of CD19 density (in patient's sample).The efficient of B cell cleaning may to mediate the ability of ADCC relevant with anti-CD 19 antibodies.Utilizing anti-CD 19 antibodies to carry out the efficient of B cell cleaning also can be relevant with host Fc γ R expression/function.

6.1 material and method

Mouse HB12a described herein and HB12b anti-CD 19 antibodies are the examples that is attached to the antibody of people CD19.Such antibody can be used for by as above 5.1 the joint described method prepare the people, humanized or chimeric anti-CD 19 antibodies.CD19 or its part for the people have mutually homospecific people, humanized or chimeric anti-CD 19 antibodies, as HB12a and HB12b antibody, can be used for the compositions and methods of the invention.Especially, have the anti-CD 19 antibodies people, humanized or chimeric in same or analogous heavy chain CDR1, CDR2 and/or CDR3 district,, can be used for the compositions and methods of the invention as HB12a or HB12b.

6.1.1 material and method

Antibody produces and sequential analysis.Produce HB 12a and HB 12b antibody in the Balb/c mouse with the immunity of mouse pre B cell system, wherein mouse pre B cell system is people such as (, MoL Cell Biol, 14:3884-94 (1994)) Zhou with the cDNA transfection of coding people CD19.Two kinds of antibody have all been delivered the 5th international symposium and the discussion of holding in Boston 3-7 day in November, 1993 about people's leukocyte differentiation antigen.

Utilize RNA to measure the application of heavy chain gene, described RNA is to use RNEASY

Small-sized test kit (QIAGEN

, Valencia is CA) from 1-5 * 10 ⁶Extract in the hybridoma.Use the SUPERSCRIPT III of 200 units

ThermoScript II, from INVITROGEN

(Carlsbad, any six aggressiveness primers of synthetic damping fluid, the 20ng of the cDNA of article one chain CA), from PROMEGA

(Madison, the RNAse inhibitor of 20 units WI) and from Denville (Metuchen, the dNTP of 80nmol NJ).The cDNA solution of 1 μ l is used as the template of heavy chain (VH) gene PCR amplification.PCR is reflected in the reaction mixture of 50 μ l amount and carries out, and described reaction mixture is by 10nM Tris-HCl (pH8.3), 5mM NH ₄Cl, 50mM KCl, 1.5mMMgCl ₂, (TaqDNA polysaccharase (Invitrogen) CA) is formed for Stratagene, LaJolla for every kind of primer of 800 μ MdNTP (Denville), 400pmol and the 10%pfu check and correction polysaccharase that comprises of 2.5U.For VL, PCR is reflected in the reaction mixture of 50 μ l amount and carries out, and described reaction mixture is by 20mM Tris-HCl (pH8.4), 50mM KCl, 1.5mM MgCl ₂, every kind of primer of 800 μ MdNTP (Denville), 400pmol and the TaqDNA polysaccharase (Invitrogen) that contains 10%pfu check and correction polysaccharase (Stratagene) of 2.5U form.After the denaturing step of 3min, 32 circulations (94 ℃ of 1min, 58 ℃ of 1min, 72 ℃ of 1min) are carried out in amplification.The back is to extend 10 minutes (Thermocycler, Perkin Elmer) down at 72 ℃.Use the foregoing 5 ' VH primer (MSVHE that mixes direction; 5 ' GGGAAT TCG AGGTGC AGC TGC AGG AGT CTGG3 ') (SEQIDNO:19) (people such as Kantor, J.Immunol.158:1175-1186 (1997)) and with C γ coding region complementary antisense primer (primer C γ 1; 5 ' GAGTTC CAG GTCACT GTC ACTGGC TCA GGGA3 ') (SEQ ID NO:20).

Utilize the cytoplasm rna that extracts to measure the application of light chain gene, described RNA extracts as described in heavy chain.Obtain 5 ' variable region nucleotide sequence from cDNA, described cDNA utilizes GeneRacer ^TMTest kit (Invitrogen) produces.With the Roll Phosphoric acid esterase with total RNA dephosphorylation.From complete full length mRNA, remove 5 ' cap sequence with tobacco acid pyrophosphatase.After mRNA is converted to cDNA, be provided for the known 5 ' primer sites of GeneRacer PCR primer, use the T4RNA ligase enzyme that GeneRacer RNA oligopolymer is connected on 5 ' end of mRNA.Use Superscript ^TMThe III RT mRNA that primer reverse transcription at random is connected with GeneRacer.Use the cDNA of special antisense 3 ' primer (GAC TGA GGC ACC TCC AGA TGT TAA CTG) (SEQ IDNO:21) first chain that increases of GeneRacer 5 ' primer (identical) and constant region with GeneRacer RNA oligopolymer.The pcr amplification that contacts to earth in 50 μ L volumes uses the TaqDNA polysaccharase (Invitrogen) of the interpolation 10%pfu check and correction polymerization (Stratagene) of damping fluid that Invitrogen recommends, 2.5U.After the 2min denaturing step, add Taq and pfu, increase with 3 steps: 94 ℃ of 30s, five circulations of 72 ℃ of 60s; 94 ℃ of 30s, five circulations of 72 ℃ of 60s; 94 ℃ of 30s, 65 ℃ of 30s, 20 circulations of 72 ℃ of 60s are extended 10min down at 72 ℃ subsequently.The Taq that adds 2.5U makes extension proceed 10min and guarantees complete 3 ' A-projection.The amplification PCR product is cloned in the pCR4-TOPO carrier, checks order and be transformed into OneShot

In the cell of TOP10 ability.As described, use PCR4-TOPO carrier special " M13 forward " and " M13 is reverse " primer each mAb light chain to be checked order from 8 clones' DNA inset for heavy chain.

After using Perkin Elmer Dye Terminator Sequencing system amplification, use ABI 377PRISM

The dna sequencing instrument is to the heavy chain and the direct and two-way order-checking of light chain PCR product of purifying, and described Perkin ElmerDye Terminator Sequencing system uses as the described AmpliTaq of light chain

Archaeal dna polymerase and the same primers as or the special primer of pCR4-TOPO carrier that are used for initial p CR amplification.The order-checking fully on both direction and antisense DNA chain to HB12a and HB12b heavy chain and light chain district.

Antibody and immunofluorescence analysis.The antigenic mono-clonal mouse of the people CD19 anti-CD 19 antibodies that is attached to used herein comprises HB12a (IgG1) and HB12b (IgG1), FMC63 (IgG2a, Chemicon International, Temecula, CA), B4 (IgG1, Beckman Coulter, Miami, FL) (people such as Nadler, J.Immunol, 131:244-250 (1983)), and HD237 (IgG2b, the 4th international symposium, vienna about people's leukocyte differentiation antigen, Austria, 1989), the isotype of HD37 antibody conversion variant people such as (, J.Immunol, 138:2793-2799 (1987)) Pezzutto.Other antibody comprises: be attached to the anti-CD19 antibody of mono-clonal mouse on the mouse CD19, MB 19-1 (IgA) (people such as Sato, JImmunol, 157:4371-4378 (1996)); Monoclonal mouse CD20 specific antibody (people such as Uchida, Intl.Immunol, 16:119-129 (2004)); B220 antibody RA3-6B2 (DNAX Corp.Palo Alto, CA); Thy1.2 antibody (CALTAG ^TMLaboratories, Burlingame, CA) and CD5, CD43 and CD25 antibody (BD PHARMINGEN ^TM, Franklin Lakes, NJ).Specific and anti-mouse Ig of isotype or IgM antibody come from Southern Biotechnology Associates, and Inc. (Birmingham, AL).

Will be with hCD19cDNA (Tedder and Isaacs, J.Immunol, 143:712-717 (1989)) or the mouse pre B cell of the leukocyte suspension transfection of individual cells be 300.19 (people such as Alt, Cell, 27:381-388 (1981)) according to the method for determining (people such as Zhou, Mol.Cell.Biol, 14:3884-3894 (1994)) use the every kind of antibody that pre-determines optimum concn to dye on ice 20-30 minute.At FACSCAN

Or FACSCALIBUR

(Becton Dickinson, San Jose have analyzed the cell with lymphocyte transhipment and sidelight scattering nature on CA) to flow cytometer.Use and the nonreactive control antibodies (CALTAG in localized path ^TMLaboratories, Burlingame CA) determines background dyeing, gets rid of＞98% cell.For the sample of every kind of detection, be used in the fluorescence intensity that shows on 40 logarithmically calibrated scales, in the case of any possible every duplicate samples analysis is had the cell of monocyte transhipment and sidelight scattering nature.

Mouse.The transgenic mice (people such as Zhou, Mol.Cell.Biol, 14:3884-3894 (1994)) for preparing expressing human CD19 (h19-1) and wild-type (WT) littermate thereof as previously mentioned.Produce the TG-1 mouse from the original h19-1 person of foundation (C57BL/6xB6/SJL), and on the C57BL/6 basis, hybridized at least for 7 generations.Produce the TG-2 mouse from the original h19-4 person of foundation (C57BL/6xB6/SJL).For after backcrossing, obtained TG-1 many ^{+ /+}Mouse, the cell surface density that people CD19 expresses on its B cell is seldom identical with the density difference of finding on people's B cell.Further described the mouse of expressing human CD19, and it has been used as model (people such as Engel, Immunity, 3:39-50 (1995) in some researchs; People such as Sato, Proc.Natl.Acad.Sci.USA, 92:11558-11562 (1995); People such as Sato, J.Immunol, 157:4371-4378 (1996); People such as Tedder, Immunity, 6:107-118 (1997); People such as Sato, J.Immunol, 158:4662-4669 (1997); People such as Sato, J.Immunol, 159:3278-3287 (1997); People such as Sato, Proc.Natl.Acad.Sci.USA, 94:13158-13162 (1997); People .J.Exp.Med.186:1923-1931 (1997) such as Inaoki; People such as Fujimoto, J.Immunol, 162:7088-7094 (1999); People such as Fujimoto, Immunity, 11:191-200 (1999); People such as Satterthwaite, Proc.NatlAcad.Sci.USA, 97:6687-6692 (2000); People such as Fujimoto, Immunity, 13:47-57 (2000); People such as Sato, J.Immunol, 165:6635-6643 (2000); People such as Zipfel, J.Immunol, 165:6872-6879 (2000); People such as Qian, J.Immunol, 166:2412-2419 (2001); People such as Hasegawa, J.Immunol, 167:2469-2478 (2001); People such as Hasegawa, J.Immunol, 167:3190-3200 (2001); People such as Fujimoto, J.Biol.Chem.276:44820-44827 (2001); People such as Fujimoto, J.Immunol, 168:5465-5476 (2002); People such as Saito, J.Clin.Invest.109:1453-1462 (2002); People such as Yazawa, Blood, 102:1374-80 (2003); People such as Shoham, J.Immunol, 171:4062-4072 (2003)).(the CD19 that CD19 lacks ^-/-) mouse and WT littermate thereof (people such as Engel, Immunity, 3:39-50 (1995)) also as previously mentioned.The endogenous mouse CD19 that expression of people CD19 in transgenic mice has been shown as lower level expresses (people such as Sato, J.Immunol, 157:4371-4378 (1996); And people such as Sato, J.Immunol, 158:4662-4669 (1997)), and hypothesis this reduction of also having determined to express about endogenic mouse CD19 people such as (, J.Immunol, 171:4062-4072 (2003)) Shoham.The density that CD19 expresses in the transgenic mice of expressing human CD19 also is (people such as Sato, J.Immunol, the 165:6635-6643 (2000)) that determines.

Use (Germantown, the mouse (FcRy of γ chain (FcR γ) defective that FcR NY) (Fc acceptor) is general from Taconic Farms ^-/-, B6.129P2-Fcerg1 ^Tml) breeding TG-1 ^{+ /+}Mouse produces hCD 19 ^+/-FcR γ ^-/-With the WT littermate.For the genetically modified mouse hemizygote of c-Myc (E μ-cMycTG, C57B1/6J-TgN (IghMyc); The JacksonLaboratory, Bar Harbor, (people such as Harris, people such as J.Exp.Med.167:353 (1988) and Adams, Nature, 318:533 (1985)) ME) as described.With c-MycTG mouse (B6/129 background) and hCD19TG-1 ^{+ /+}Mouse hybridizes and produces hemizygous hCD 19TG-1 ^+/-CMycTG ^+/-The offspring is as determined by the PCR screening.Rag1 ^-/-(B6.129S7-Rag1 ^TmlMon/ J) mouse comes from Jackson Laboratory.According to standard method (Van Rooijen and Sanders, J.Immunol.Methods, 174:83-93 (1994)), (0.1mL/10 restrains body weight to the liposome of clodronate disodium being sealed at the 2nd, 1 and 4 day; SigmaChemical Co.St.Louis MO) is expelled to by tail vein and produces scavenger cell defective mouse in the C57BL/6 mouse.The AU mouse is placed in the device of specific pathogen-free domestic obstacle, and uses first during age in week at 6-9.

ELISAs。By using affinity purifying mouse IgM, IgG1, IgG2a, IgG2b, IgG3 and IgA (Southern Biotechnology Associates, Inc.) ELISA produces described typical curve (people such as Engel, Immunity, 3:39 (1995)), determine serum I g concentration.ELISA by the bovine chest gland DNA (it comprises single stranded DNA) that uses described ox thymus dsdna (Sigma-Aldrich), boil or the microtiter plate (people such as Sato, J.Immunol.157:4371 (1996)) that scribbles histone (Sigma-Aldrich) determines serum IgM and the IgG autoantibody level with respect to dsDNA, ssDNA and histone.

Immunotherapy.Be injected at anti-CD19 of disinfectant and nullvalent isotype control antibodies (0.5-250 μ g) in the 200 μ L phosphate buffered saline (PBS)s (PBS) by edgewise tail vein.The antibody of 250 μ g is used in whole experiments, unless indicated other.Behind globulolysis, measure the number of blood leucocyte by hematimeter, determine B220 by immunofluorescence dyeing with stream cell counting analysis ⁺The frequency of B cell.Use Oncology Tool DoseCalculator (www.fda.gov/cder/cancer/animalframe.htm) comes the antibody dosage in comparison people and the mouse.

Immunization method.With 50 μ g2 in the salt solution, 4,6-trinitrophenyl (TNP) bonded lipopolysaccharides (LPS) (Sigma, St.Louis, MO) or 25 μ g2,4-dinitrophenol bonded (DNP)-FICOLL (Biosearch Technologies, San Rafael, CA) two months big wild-type (WT) mouse of intraperitoneal (i.p.) immunity.Also be used in complete Freund's adjuvant (100 μ gDNP bonded key hole keyhole limpet hemocyanin (DNP-KLH, the CALB IOCHEMs of complete Freund ' in sadjuvant)

-NOVABIOCHEM

Corp.La Jolla, CA) immune mouse, and after 21 days, be used in DNP-KLH reinforced immunological in the incomplete Freund's adjuvant (incomplete Freund ' s adjuvant).As noted, before and after immunity, mouse is got blood.According to standard method (people such as Engel, Immunity, 3:39-50 (1995)) with scribbling DNP-BSA (CALBIOCHEM -NOVABIOCHEM

Corp.La Jolla, CA) or TNP-BSA (elisa plate CA) is determined at DNP-or TNP-specific antibody titre in the single serum sample in copy for Biosearch Technologies, SanRafael.To dilute 1: 400 from the serum of TNP-LPS mice immunized, will be from DNP-FICOLL

Diluted 1: 1000 with the serum of DNP-BSA mice immunized, be used for elisa assay.

Tumor research.Will be from hCD19TG-1 ^+/-c-mycTG ^+/The lymphoglandula tumour that exists naturally of-mouse is separated, and amplification in vivo.At the 0th day with tumour cell (10 ^-5/ mouse) gives Rag-A by i.v. and accept mouse, and FMC63 and pairing isotype contrast mAbs (250 μ g/ml) are passed through the i.v. administration at the 1st and 7 day.To separate from the blood leucocyte of accepting mouse weekly, and analyze definite round-robin mouse CD19 by immunofluorescence dyeing and stream cell counting ⁺B220 ⁺The number of cell.

Statistical analysis.Whole data are expressed as mean value ± SEM.T with the student examines and determine the difference significance of determining between sample mean.

6.2 embodiment 1: the expression of people CD19 in transgenic mice

The transgenic animal of transgenosis hCD19TG mouse described herein or other expressing human CD19 can be used to estimate the different treatment plans that comprise the anti-CD 19 antibodies people, humanized or chimeric, for example in drug concentration, total amount or change on the time.Effectiveness at the human patients of different treatment plans can utilize two kinds of indexs as described below to predict, that is: the B cell consumption in some body fluid and/or tissue and monoclonal the people's or humanized anti-CD 19 antibodies be attached to ability on the B cell.In specific embodiment, effective treatment plan and the compositions and methods of the invention can be used for treating the B cell malignancies in the human body in people CD19 transgenic mice.

In order to determine that people CD19 is whether from transgenic mice (hemizygous TG-1 ^+/-) the B cell on express, the B cell is extracted from marrow, blood, spleen and the seroperitoneum of these mouse.By cell is contacted the expression of appraiser CD19 and mouse CD19 in these cells with mono-clonal anti-CD 19 antibodies in conjunction with the mouse of CD19.Utilize double-colored immunofluorescence dyeing, measuring antibody by stream cell counting analysis is the combination of cell for B.

The result is presented at Figure 1A, has promptly shown the figure of the expression (X-axis) of the mouse CD19 (mCD19) that measures to the curve of the expression of people's CD19 (hCD19) (Y-axis) in marrow (BM), blood, spleen and the seroperitoneum (PL) measured.The unit representation of axle is from 1 40 logarithmically calibrated scales that begin on the lower left side.The B4 anti-CD 19 antibodies that will be attached to people CD19 (Beckman/Coulter) is used to make the expression of people CD19 to specialize, and the 1D3CD19 antibody that will be attached to mouse CD19 (PharMingen) is used to make the expression of mouse CD19 to specialize (also being used for Figure 1B and 1C).When increasing people CD19 expression gradually during people's B cell development, mouse CD19 is at high level expression during mouse bone marrow cells B cell development.Figure 1A has shown that the expression of people CD19 is parallel to the expression of mouse CD19 in the periphery B cell that obtains in blood, spleen and seroperitoneum (PL), show that mouse anti hCD19 antibody (it is in conjunction with people CD19) is in conjunction with periphery B cell mass.In addition, derive endogenous mouse CD19 of B cell expressing rather than people CD19 (being attached to the mono-clonal mouse anti-CD 19 antibodies of people CD19) of Xiao Liang marrow (BM).Therefore, at hemizygous TG-1 ^+/-Marrow B cell is divided into two classes in the mouse: sophisticated B is a cell, i.e. hCD19 ⁺MCD19 ⁺With less sophisticated B be cell, i.e. mCD19 only ⁺(Figure 1A).These results are consistent with people's (Mol.Cell.Biol.14:3884-3894 (1994)) such as Zhou discovery, and described document points out that the expression of people CD19 in these transgenic mices is relevant with the maturation of B cell.All mature B cells in blood, spleen and the peritoneal cavity all are hCD19 ⁺And mCD19 ⁺

Relative expression's level of mCD19 and hCD19 as estimating by measuring average fluorescent strength (mouse is to the anti-CD19 of hCD19 and the mouse anti-CD19 to mCD19) respectively, is shown among Figure 1B.TG-1 mouse (the TG-1 that is isozygotying for the hCD19 transgenosis ^+/-) in, the hCD19 on the B cell that blood produces expresses to express with the hCD19 on human B cell and compares.In order to compare at TG-1 ^{+ /+}, TG-1 ^+/-, TG-2 ^{+ /+}The relative density that hCD19 and mCD19 express in the transgenic mice system has been extracted blood deutero-B cell, and has been measured the expression of CD19 as mentioned above.The result is presented among Figure 1B with histogram, and the human blood B cell that it has shown expressing human CD19 accounts for the TG-1 from the hCD19TG mouse ^{+ /+}, TG-1 ^+/-And TG-2 ^{+ /+}The per-cent (left side) of blood B cell, and wild-type (WT) the mouse blood B cell of expressing mouse CD19 account for the TG-1 from the hCD19TG mouse ^{+ /+}, TG-1 ^+/-And TG-2 ^{+ /+}CD19 ⁺The per-cent (right side) of blood B cell.This value (linear value of average fluorescent strength) expression CD19 express with the average relative density of comparing from people or wild-type (WT) mouse (being shown as 100%) (± SEM).The result shows the TG-1 that is isozygotying ^{+ /+}In the mouse, for the density of measuring with average fluorescent strength, the blood B cell of expressing hCD19 is than human blood B cell high about 72%.At the TG-1 that expresses hCD 19 ^+/-Blood B cell is similar to human blood B cell on density in the mouse, and at the TG-2 that expresses hCD 19 ^{+ /+}Blood B cell hangs down 65% than human blood B cell in the mouse on density.

In Fig. 1 C, shown from TG-1 with histogram ^+/-The further comparison of the relative density that hCD19 in the B cell of mouse tissue and mCD19 express, it shows the B cell dyeing from marrow, blood, spleen, lymphoglandula and PL, the average fluorescent strength of anti-CD 19 antibodies (MFI ± SEM), wherein hCD19 (left side) and mCD19 (right side).The result shows at TG-1 ^+/-In the mouse, hCD19 is by the B220 in marrow ⁺Cell the horizontal expression that improves (human blood level 63%)＜blood (100%)＜spleen (121%)=lymphoglandula (120%) also＜peritoneal cavity (177%).People CD19 expression is expressed mCD19 less influence.The level of the mRNA of hCD19 and mCD19 does not change.

For the mouse anti hCD19 antibody (it is attached on the people CD19) of determining IgG1 (HB 12a, HB12b, B4), IgG2a (FMC63) and IgG2b (HD237) isotype work whether variant, with the B220 of blood and spleen ⁺The B cell is from TG-1 ^+/-Extract in the mouse.The cell that extracts is contacted at external and above anti-CD 19 antibodies, and, estimate its ability in conjunction with people CD19 express transgenic mouse (hCD19TG) B cell by the concrete monoclonal antibody dyeing of using isotype specificity PE conjugated secondary antibodies and stream cell counting to analyze.

In Fig. 1 D, IgG2b (isotype of mouse), the IgG2a (isotype of mouse) of 5 μ g/ml and the figure of the relative B cell number (Y-axis) of IgG1 (isotype of mouse) anti-CD 19 antibodies have been shown the result with fluorescence intensity (X-axis).Painted B220 ⁺The fluorescence intensity and the anti-CD 19 antibodies of cell are shown as solid line, and the fluorescence intensity of paired isotype contrast (CTL) is shown as dotted line.Each antibody is issued to saturated level with spleen B cell response in the concentration of 5 μ g/ml.The result shows from TG-1 ^+/-Mouse blood of mouse and spleen B220 ⁺Anti-CD 19 antibodies on the B cell is identical in conjunction with density for the antibody isotype of testing, and all like this for blood and spleen B cell.

In order to determine whether average fluorescent strength is irrelevant with the anti-CD 19 antibodies isotype, by dyeing mouse pre B cell is 300.19, utilize identical anti-mouse Ig secondary antibody to carry out transfection with hCD19cDNA, the combination of estimating independently anti-CD 19 antibody (at 5 μ g/mL) is active.By stream cell counting analysis and utilization mouse Ig specificity PE-bonded secondary antibody, measure antibody staining (MFI ± SEM).In Fig. 1 E, shown the result with histogram, described histogram is for HB 12a, HB 12b, B4, FMC63, HD237 anti-CD 19 antibodies and control antibodies (CTL), anti-CD 19 antibodies is in conjunction with (as directed by staining power, the Y-axis) histogram to 300.19 cells of hCD19cDNA transfection.Be independent of each antibody staining cell with characteristic average fluorescent strength of anti-CD 19 antibodies isotype, HB12b has shown the dyeing of minimum level, and HD237 has showed the dyeing of highest level.Therefore, result displayed shows that 300.19 cells are to be used for the model of comparison anti-CD 19 antibodies in the vitro system of the ability of the external CD19 of being attached to.

Therefore, altogether, result shown in Figure 1 shows that the representative of hCD19TG mouse and 300.19 cells is used for when hCD19 expresses in density range necessarily, estimates the suitable external and body inner model system of the ability of anti-hCD19 antibodies to the B cell.

Figure 1A-D represents the result that obtains by in the mouse of every kind of genotype＞3.

6.3 embodiment 2: the anti-CD 19 antibodies consumption of B cell in the body

Mouse anti-CD 19 antibodies (it is attached to people CD19) has been estimated its body internal consumption hCD19TG (TG-1 ^+/-) ability of blood, spleen and lymphoglandula B cell.Give mouse with each antibody with the amounts of 250 or 50 μ g/ mouse, single dosage than the anti-CD20 treatment of human body basically with low about 10 to 50 times of the dosage of four administration 375mg/m2 (people such as Maloney, J.Clin.Oncol, people such as 15:3266-74 (1997) and McLaughlin, 12:1763-9 (1998)).

In Fig. 2 A, shown the result with the piece figure that carries out CD19 or the B cell of paired isotype contrast (CTL) treatment after 7 days with HB12a, HB12b or FMC63 anti-CD 19 antibodies or contrast.For lymphoglandula, spleen and blood tissues for each anti-CD 19 antibodies or contrast provide isolating.At the per-cent that the passage lymphocyte in 7 days shown in every consumes, show by immunofluorescence dyeing and stream cell counting analyze determine from TG-1 ^+/-The typical B cell consumption of the blood of mouse, spleen and lymphoglandula.Fig. 2 B is presented at after anti-CD19 (closed circle) or isotype contrast (open circle) Antybody therapy, B220 ⁺The average number of blood B cell (every ml ± SEM).The data that value representative shown in after the time 0 obtained at 1 hour.Fig. 2 C and Fig. 2 D are presented at anti-CD19 (band of filling) or contrast (open band) the antibody treatment TG-1 with specified dosage respectively ^+/-After the mouse, the number of spleen and lymphoglandula B cell (± SEM).In Fig. 2 B-D, shown with the significant difference between the average result of the mouse (each data point＞3 mouse) of anti-CD19 or isotype control antibodies treatment; Compared with the control, ^*P＜0.05, ^*P＜0.01.

Most of circulation B cells (Fig. 2 B) of each antibody consumption within a hour of treatment had strong consumption effect for spleen and lymphoglandula B cell frequency (Fig. 2 A) and number (Fig. 2 C-D) in seven days.In seven days HB 12a antibody consumption 98% blood B cell, the spleen of 90-95% and lymphoglandula B cell.Similarly, HB12b, B4, FMC63 and HD237 antibody consume 99%, 96%, 99% and 97% blood B cell respectively.HB12b, B4, FMC63 and HD237 antibody consume spleen and the lymphoglandula B cell of 88-93%, 64-85%, 72-95% and 88-90% respectively.The basic previous generation of the remaining periphery B cell of minority has shown immature cell on the phenotype, and it shifts from marrow potentially.When to the administration of WT mouse, CD19 antibody does not have tangible effect, and the paired isotype control antibodies that gives does not under the same conditions influence B cell number (Fig. 2 A-D).Therefore, consumed B cell effectively by seven days anti-hCD19 antibody from circulation, spleen and the lymphoglandula of hCD19TG mouse.Will be at TG-1 ^+/-The general introduction of B cell consumption is provided in the table 1 in the mouse.

Table 1

^aThe b cell subsets is: marrow (BM) pro B lymphocyte (CD43 ⁺IgM-B220 ^Lo), pre B cell (CD43 ^-IgM ^-B220 ^Lo), immature B cell (IgM ⁺B220 ^Lo), sophisticated B cell (IgM ⁺B220 ^Hi); Abdomen B1a (CD5 ⁺B220 ^Lo), B2 (CD5 ^-B220 ^Hi).

^bValue (± SEM) show at Antybody therapy (250 μ g) and be present in the cell number (x10 in the mouse in seven days afterwards ^-6).The BM value is for the bilateral femur.The blood number is the number of each ml.The LN number is nodular for bilateral inguinal region and armpit.The mouse number is indicated in bracket.Pointed out the significant difference between the mean value; ^*P＜0.05, ^*P＜0.01.

6.3.1 the consumption of marrow B cell

In the hCD19TG mouse, measured known anti-CD 19 antibodies, determined whether such antibody is effective in the B cell that consumes from various body fluid and tissue.Test described herein can be used for determining other anti-CD 19 antibodies, for example, is attached to the anti-CD 19 antibodies of the antigenic specific part of people CD19, whether can consume the B cell effectively.Use is defined as consuming the result of the anti-CD 19 antibodies of B cell can be relevant with the application in human body.Antibody with characteristic of definite antibody can be used to human body B cell malignancies therapeutic composition and the method for being used for of the present invention.Fig. 3 A-3F has described the marrow B cell consumption according to the CD19 Antybody therapy.

Fig. 3 A has shown the figure of fluorescence intensity (X-axis) to relative B cell number (Y-axis), wherein four look immunofluorescence dyeings by cell, stream cell counting analysis and lymphocytic forward with side dispersive character, estimate by TG-1 ^+/-HCD19 that marrow B cell subtype carries out and the expression of mCD19.Pro B lymphocyte is defined as CD43 ⁺IgM ^-B220 ^Lo, pre B cell is CD43 ^-IgM ^-B220 ^Lo, immature B cell is IgM ⁺B220 ^Lo, sophisticated B cell is IgM ⁺B220 ^HiBar graph (right side) shows the average relatively MFI (± SEM) value of CD19 that each B cell subsets (each data point＞3 mouse) is expressed.As in the hCD19TG mouse (Figure 1A), it is uneven that CD19 is expressed in the human body, because B cell maturation and break away from marrow.Only a fraction of pro B lymphocyte (20%, CD43 ^HiIgM ^-B220 ^Lo) at TG-1 ^+/-Express hCD19 in the mouse, and most pre B cell is hCD19 ⁺, and the most of mature B cells in marrow are with than higher horizontal expression hCD19.The half pro B lymphocyte (55%, IgM ^-B220 ⁺) at TG-1 ^+/-Express mCD19 in the mouse, and mCD19 by most of pre B cells and mature B cell in marrow with than higher horizontal expression.

Fig. 3 B is presented at FMC63 or paired isotype control antibodies (250 μ g) was treated back seven days, by double-colored immunofluorescence dyeing and stream cell counting assay hCD19 in the hCD19TG mouse ⁺The consumption of cell.The number representative is in the relative frequency of specified route inner cell.The result shows be the genotypic three brood births of each mouse to obtain.According to the CD19 Antybody therapy, the FMC63 antibody by giving with 250 μ g/ mouse has consumed at TG-1 ^{+ /+}, TG-1 ^+/-And TG-2 ^{+ /+}Most of hCD19 in the marrow of mouse ⁺Cell.

Fig. 3 C has shown at TG-1 ^+/-Anti-CD19 of mouse or pairing isotype control antibodies (250 μ g) treatment seven days afterwards, typical B 220 ⁺The B cell consumption.B220 in the antibody of value representative femur of bilateral in the mouse of treatment of bar graph ⁺The sum of cell (± SEM).Pointed out the significant difference between sample mean (every group＞3 mouse); ^*P＜0.05, ^*P＜0.01.Unexpectedly, from marrow, also consume most mCD19 at low expression hCD19 on undetectable level ⁺Pre B cell.Therewith as one man, the most marrow B220 of FMC63, HB12a, HB12b, B4 and HD237 antibody consumption ⁺Cell.

Fig. 3 D is presented at TG-1 ^+/-The consumption of back seven days typical marrow B cell subsets of FMC63 of mouse or paired isotype control antibodies (250 μ g) treatment, it is estimated by the trichromatic immunofluorescence dyeing.According to the expression (figure of below) of CD43, with IgM ^-B220 ^LoFormer/pre B cell further segments.Fig. 3 E is presented at FMC63 or back seven days typical marrow CD25 of paired isotype control antibodies (250 μ g) treatment of hCD19TG mouse system ⁺B22 ^LoThe consumption of pre B cell, it is estimated by double-colored immunofluorescence dyeing.The result comes from the experiment of carrying out in different skies, so passage is not determined.When having analyzed one marrow subgroup, most CD43 ^HiIgM-B220 ^LoPro B lymphocyte (Fig. 3 D) is not subjected at TG-1 ^{+ /+}, TG-1 ^+/-Or TG-2 ^{+ /+}The influence of FMC63 Antybody therapy in the mouse, and consumed most CD25 ⁺CD43 ^LoIgM ^-B220 ^LoPre B cell (Fig. 3 E).Fig. 3 F is presented at＞Antybody therapy of the right FMC63 (sealing band) of 3 brood births or contrast (open band) after seven days pro B lymphocytes in the femur of bilateral, pre B cell, immature and sophisticated B cell number (± SEM).The result shows also and has consumed from TG-1 ^{+ /+}, TG-1 ^+/-Or TG-2 ^{+ /+}The most of immature and sophisticated B cell of the marrow of mouse.Therefore, consumed myeloid most of hCD19 by the CD19 Antybody therapy ⁺Cell is included in the pre B cell of low expression level hCD19.

6.3.2 the consumption of belly B cell

At TG-1 ^+/-Peritoneal cavity B cell and other of mouse organizes the B cell to compare, and expresses hCD19 (Figure 1A and Fig. 1 C) on higher level, mainly due to CD5 ⁺IgM ^HiB22O ^LoThe existence of B1 cell, itself and the CD5 of commonly used (B2) B cell ^-IgM ^LoB220 ^HiHypotype is compared, and expresses high about 25% (Fig. 4 A) of hCD19.Fig. 4 B-4C shows peritoneal cavity B cell antagonism CD19 Antybody therapy sensitivity.

Fig. 4 A shows that the expression (X-axis) of human and mouse CD19 is to peritoneal cavity CD5 ⁺B220 ⁺BIa and CD5 ^-B220 ^HiThe figure of the relative number (Y-axis) of B2 (commonly used) B cell.With three look immunofluorescence dyeings and the lymphocytic individual cells suspension of flow cytometry analyzing and testing peritoneal cavity.Bar chart is represented with 3 couples of littermate TG-1 ^+/-The MFI that the CD19 of mouse expresses (± SEM) mean value.

Fig. 4 B shows with CD19 (HB12a of 250 μ g, HB12b and FMC63); The B4 of 50 μ g and HD237 antibody or control antibodies (250 μ g) handle from TG-1 ^+/-The peritoneal cavity B220 of mouse ⁺The consumption of cell.Numerical statement is shown in B220 in the path of pointing out in the 7th day ⁺The correlated frequency of cell.The value representation of bar chart is at the intraperitoneal B220 of the mouse (every group＞3 mouse) of Antybody therapy ⁺The sum of cell (± SEM).Point out the significant difference between sample mean; ^*P＜0.05, ^*P＜0.01.The result shows the 7th day, and the treatment of anti-CD 19 antibodies has consumed most belly B220 in 250 μ g/ mouse ⁺The B cell.Result displayed is partly explained by B1 and B2 cell commonly used in Fig. 4 B.When at TG-1 ^{+ /+}When expressing hCD19 with the highest density in the mouse, most B1 and B2 cell have been consumed.Yet when the hCD19 level was low, the consumption of the CD19 mediation of B1 and B2 cell was at TG-1 ^+/-And TG-2 ^{+ /+}It in the mouse poor efficiency.Therefore, the density that relies on its CD19 that utilizes average fluorescent strength to measure to express, the CD19 Antybody therapy consumes the B1 and the B2 cell of belly, has more resistance though belly B cell and spleen and lymphoglandula B cell compare the consumption of anti-CD 19 antibodies mediation.

Fig. 4 C is presented at anti-CD 19 antibodies or the control antibodies of hCD19TG mouse and treated CD5 seven days afterwards ⁺B220 ⁺B1a and CD5 ^-B220 ^HiThe typical case of B2B cell consumes.Numerical statement is shown in the correlated frequency of every kind of B cell subsets in the specified path.The value representation of bar chart the sum of every kind of B cell subsets of intraperitoneal of the mouse (every group＞3 mouse) of Antybody therapy (± SEM).Point out the significant difference between sample mean; ^*P＜0.05, ^*P＜0.01.

6.3.3 the B cell of isolating anti-CD 19 antibodies mediation is removed

Whether different in order to measure HB12a with known anti-CD 19 antibodies with the HB12b anti-CD 19 antibodies, analyzed each anti-CD 19 antibodies amino acid sequences (Fig. 5 A and 5B, 6A and 6B, 7A and 7B) used herein.

Fig. 5 A describes the Nucleotide (SEQ ID NO:1) of the heavy chain VR-D-JH catenation sequence of HB12a anti-CD 19 antibodies and amino acid (the SEQ ID NO:2) sequence of prediction.Point out and the corresponding to sequence of 5 ' PCR primer that by double underline it can be different from actual dna sequence dna, because used unnecessary primer.In sequence, specify in approximate fillet between V, D and the J sequence by vertical band (1).The Nucleotide password points out that Nucleotide is attached on the potential site of fillet or the super sudden change of body in the minority case.The n terminal residue (E) of antibody is labeled as residue 1.

Fig. 5 B describes the Nucleotide (SEQ ID NO:3) of the heavy chain VH-D-JH catenation sequence of HB12b anti-CD 19 antibodies and amino acid (the SEQ ID NO:4) sequence of prediction.Point out and the corresponding to sequence of 5 ' PCR primer that by double underline it can be different from actual dna sequence dna, because used unnecessary primer.In sequence, specify in approximate fillet between V, D and the J sequence by vertical band (1).The Nucleotide password points out that Nucleotide is attached on the potential site of fillet or the super sudden change of body in the minority case.The n terminal residue (E) of antibody is labeled as residue 1.

Fig. 6 A describes the Nucleotide (SEQ ID NO:15) of the light chain VK-JK catenation sequence of HB12a anti-CD 19 antibodies and amino acid (the SEQ ID NO:16) sequence of prediction.Fig. 6 B describes the Nucleotide (SEQ ID NO:17) of the light chain V-J catenation sequence of HB12b anti-CD 19 antibodies and amino acid (the SEQ ID NO:18) sequence of prediction.It is No. 1 that the amino terminal amino acid of the ripe secretory protein that will be derived by amino acid sequence analysis is compiled.Point out and the corresponding to sequence of 3 ' PCR primer by double underline.Pointed out the fillet (/) of the prediction in V-J-C district, the J district Nucleotide of runic is represented the potential site of the super sudden change of body.

Fig. 7 A and 7B describe the aminoacid sequence comparison of disclosed mouse anti-CD 19 antibodies.Fig. 7 A shows the sequence alignment of the heavy chain VR-D-JH catenation sequence that comprises consensus sequence (SEQ ID NO:5), HB12a (SEQ ID NO:2), 4G7 (SEQ ID NO:6), HB 12b (SEQ ID NO:4), HD37 (SEQ ID NO:7), B43 (SEQ ID NO:8) and FMC63 (SEQ ID NO:9).According to ordinary method (people such as Kabat, Sequences of Proteinsof Immunological Interest.U.S.Government Printing Office, Bethesda, MD (1991)), come amino acid is numbered and specified the initial point of the encoding sequence in each antibody V, D and J district, wherein by VH gene coding amino acid site 1-94 and complementarity-determining region CDR1 and 2.The breach of dotted line indication insertion sequence maximizes the comparison of similar aminoacid sequence.The identity of round dot indication between the sympathetic aminoacid sequence of each anti-CD 19 antibodies and all antibody.Outstanding CDR district makes it clear.Fig. 7 B shows the analysis of the light chain VK aminoacid sequence of anti-CD 19 antibodies.Consensus sequence (SEQ ID NO:10), HB 12a (SEQ ID NO:16), HB 12b (SEQ ID NO:18), HD37 (SEQ ID NO:11), B43 (SEQID NO:12), FMC63 (SEQ ID NO:13) and 4G7 (SEQ ID NO:14) have been listed.According to ordinary method (people such as Kabat, Sequences of Proteins of ImmunologicalInterest.U.S.Government Printing Office, Bethesda, MD (1991)), come amino acid is numbered and specified the initial point of the encoding sequence of each anti-CD 19 antibodies, wherein by VH gene coding amino acid site 1-94 and complementarity-determining region CDR1 and 2.To compile according to the amino acid of the signal sequence cracking site of predicting is No. 1.The breach of dotted line indication insertion sequence maximizes the comparison of similar aminoacid sequence.Outstanding CDR district (square frame) makes it clear.

Because each anti-CD 19 antibodies of being measured in this research has consumed the interior B cell of the body of important number, estimate each anti-CD 19 antibodies amino acid sequences and determine whether these antibody are different on sequence, and whether can be incorporated on the different CD19 epi-positions.Antibody is by molecular interaction, and promptly the effect that is mediated by specific amino acid in the variable region of each antibody molecule is in conjunction with target antigen.Therefore, for each antibody and its special aminoacid sequence, almost be unique with the antibody that is attached to the special epi-position on these antigen in the complex interactions between the proteantigen.The level of this complicacy in antigen and antibody interaction is the reflections of various antibody all constituents for most of proteantigens.When antibody and target antigen interact mainly is when being mediated by the amino acid within the complementarity-determining region (CDR) at antibody molecule, and skeleton amino acid also is conclusive for antigen-binding activity.Therefore, similar antibody is attached on the same antigen or zone of target molecule probably on the structure, and has on the structure in different V and CDR district dissimilar antibody probably by different molecular interactions and antigenic different regional interaction.

Because interact to the same molecular zone (or epi-position) of target antigen and bonded antibody according to similar on the definition structure, the aminoacid sequence that has compared HB12a, HB12b, FMC63 and other disclosed anti-CD 19 antibodies, comprise: HD37 (people such as Kipriyanov, J.Immunol.Methods, 196:51-62 (1996); People such as Le Gall, FEBSLetters, 453:164-168 (1999)), 2G7 (people such as Meeker, Hybridoma, 3:305-320 (1984); People such as Brandl, Exp.Hematol, 27:1264-1270 (1999)) and B43 (people such as Bejcek, Cancer Res.55:2346-2351 (1995)) antibody.By V (D) J gene fragment and the V district that derives from V1S39, V1S56, V1S136 or V2S1 gene fragment, derive from the D district of FL16.1 gene fragment and derive from J2 or the various combination in the J district of J4 gene fragment produces the heavy chain (table 2) of anti-CD 19 antibodies.B43 comes down to identical (Fig. 7 A-B) with disclosed heavy chain of HD37 antibody and variable region of light chain on aminoacid sequence.The maintenance of this level has reflected such fact: each in these antibody is also obviously similar on nucleotide level, have being connected of identical VH (D) JH and VLJL, come each cDNA sequence of pcr amplification to calculate most difference by utilizing unnecessary primer.This shows that HD37 and B43 and antibody share common, though origin inequality, and therefore be attached on the proteic identical epi-position of CD19.HB12a and 4G7 antibody also are different from other anti-CD 19 antibodies.Though the heavy chain district of HB12a and 4G7 antibody is similar, and may derive from identical embryonal system VH (D) JH gene fragment, different joint borders is used for the assembling (Fig. 7 A) of D-JH.HB12b and 4G7 antibody utilize isolating VH gene fragment (table 2), and have and the visibly different CDR3 sequence of another anti-CD 19 antibodies (Fig. 7 A).FMC63 antibody also has and the diverse aminoacid sequence of another anti-CD 19 antibodies.

Table 2

N.D. do not detect.

^aThe number of the nucleotide difference of the numeral in bracket between CD19 antibody coding gene and the homologous embryonal system sequence determined in the database has at present been got rid of and PCR primer overlapping areas.

^bThe GENBANK of gene order

Accession number.

Shown in Fig. 7 B, each HB12a, HB12b, FMC63,4G7 and HD37/B43 antibody utilize different light chain gene (Fig. 7 B).Produce light chain from many times V and J gene fragment.The shortage of homogeneity shows that these antibodies are to some different sites of people CD19 in these six anti-CD 19 antibodies H and L chain-ordering.The aminoacid sequence of paired heavy chain and light chain show that more further most of these anti-CD 19 antibodies are structurally different, and can thus by different interactions of molecules in conjunction with people CD19.Therefore, the ability that anti-CD 19 antibodies consumes the B cell in vivo is not limited on same loci the limited antibody in conjunction with CD19, but anti-CD 19 antibodies is as the general property of a class.

6.3.4CD19 density influences the validity of the B cell consumption of CD19 antibody induction

In order to determine whether the ability that anti-CD 19 antibodies consumes the B cell depends on CD19 density, HB12b and FMC63 anti-CD 19 antibodies had the mouse that the CD19 of different levels expresses.The result shows that when anti-CD 19 antibodies exists people CD19 density and antibody isotype can influence the consumption of B cell on the B cell.Identical test can be used for determining whether other anti-CD 19 antibodies can consume the B cell effectively, and the treatment of the human patients that can express with the CD19 with different levels of result is relevant.Therefore, check that the testee CD19 exists and the method for density can be used for identifying patient or patient crowd in being used for described in the 5.5.3 joint, wherein some anti-CD 19 antibodies can consume the B cell, and/or is used for determining proper dosage.

Though more than the result of Cun Zaiing shows when when 250 or 50 μ g use, at TG-1 ^+/-Whole five kinds of anti-CD 19 antibodies of measuring in the mouse are effectively same, but for the B cell that comes autoblood, marrow and spleen, it is relevant with antibody isotype that the degree of B cell consumption appears, IgG2a＞IgG1＞IgG2b (Fig. 2 A-2D).Therefore, compared at the TG-1 that isozygotys that expresses CD19 with different density ^{+ /+}, heterozygosis TG-1 ^+/-With the TG-2 that isozygotys ^{+ /+}The efficient of HB12b in the mouse (IgG1) and FMC63 (IgG2a) antibody (Figure 1A-E).

In order to determine whether CD19 density influences the efficient of anti-CD 19 antibodies inductive B cell consumption, in the hCD19TG mouse, measure the B cell consumption of typical blood and spleen afterwards at HB12b (Fig. 8 A) or FMC63 (Fig. 8 B) Antybody therapy (7 days, 250 μ g/ mouse).The B220 in numeral path ⁺Lymphocytic per-cent.Strip chart be shown in the number of blood (every mL) or spleen (sum) B cell after anti-CD 19 antibodies (closed band) or isotype contrast (open band) treatment (± SEM).Shown the significant difference between the average result in the mouse (each data point＞3 mouse) of anti-CD 19 antibodies or the treatment of isotype control antibodies; ^*P＜0.05, ^*P＜0.01.

The result who is present in Fig. 8 A-8D shows that CD19 density influences the efficient of B cell consumption in vivo by anti-CD 19 antibodies.At the 7th day, at TG-2 ^{+ /+}Low-level CD19 expresses with the circulation of HB12b antibody or organize the consumption of B cell that remarkable influence (Fig. 8 A) is arranged in the mouse.By TG-1 ^{+ /+}, TG-1 ^+/-And TG-2 ^{+ /+}Difference during the CD19 of mouse expresses also influences with the circulation of FMC63 antibody and organizes the consumption of B cell, and the consumption (Fig. 8 B) of not obvious change circulation B cell.

In order further to confirm that CD19 density is an important factor in the B cell consumption of CD19mAb mediation, has directly compared CD19TG-1 ^{+ /+}And CD19TG-2 ^{+ /+}The relative consumption ratio.By mark from hCD19TG-1 ^{+ /+}And CD19TG-2 ^{+ /+}The no function splenocyte of mouse comes the difference mark from CD19TG-1 ^{+ /+}And CD19TG-2 ^{+ /+}The splenocyte of mouse is used 0.1 and 0.01 μ MVybrant respectively according to the specification sheets of product ^TMCFDASE (CFSE; Molecular Probes) mark is from hCD19TG-1 ^{+ /+}And CD19TG-2 ^{+ /+}The no function splenocyte of mouse.Determine B220 in the splenocyte of CFSE mark by immunofluorescence dyeing and stream cell counting analysis ⁺The relative frequency of cell.Afterwards, with the B220 of the CFSE mark of equal number ⁺HCD19TG-1 ^{+ /+}With hCD 19TG-2 ^{+ /+}Splenocyte (2.5 * 10 ⁵) be expelled in the peritoneal cavity of three kinds of wild-type B6 mouse.After 1 hour, give mouse FMC63 or the contrast rnAb (250 μ g, i.p.).After 24 hours, reclaim the lymphocyte of mark, by the B220 of stream cell counting measuring CFSE mark ⁺And B220 ^-The correlated frequency of cell.Path in each column diagram of Fig. 8 C is presented at CD19TG-1 ^{+ /+}(CFSE ^High) and CD19TG-2 ^{+ /+}(CFSE ^Low) the splenocyte group in B220 ⁺The frequency of cell.Histogram represents to be present in the number of the mouse of anti-CD19mAb treatment with respect to the cell mass of the CFSE mark in the mouse of contrast mAb treatment.The result represents to transfer to＞and 3 wild-types accept the hCD 19TG-1 in the mouse ^{+ /+}Splenocyte (band of filling) and hCD19TG-2 ^{+ /+}Splenocyte (open band), the sample mean of indication (± there were significant differences between SEM); ^*P＜0.01.

After anti-CD19 or contrast mAb treat one mouse 24 hours, measure the removing of B cell.Compare with the mouse of the mAb treatment of contrast, with than the CD19TG-2 in the mouse of anti-CD19mAb treatment ^{+ /+}The obvious faster rate of B cell (p＜0.01) consumes CD19TG-1 ^{+ /+}B220 ⁺B cell (Fig. 8 C).In addition, CD 19TG-1 in the mouse of anti-CD19mAb treatment ^{+ /+}B220 ⁺The B cell is to CD19TG-2 ^{+ /+}B220 ⁺The relative frequency of B cell is starkly lower than (p＜0.01) CD19TG-1 in the mouse of contrast mAb treatment ^{+ /+}B220 ⁺The B cell is to CD19TG-2 ^{+ /+}B220 ⁺The ratio of B cell.Similarly, also can be relatively in anti-CD19 or contrast mAb mouse CD19TG-1 ^{+ /+}And CD19TG-2 ^{+ /+}The B220 of CFSE mark ^-The number of cell.Therefore, than the TG-2 that expresses CD 19 with low density ^{+ /+}The faster ratio of B cell ground consumes the CD19TG-1 that expresses high-density CD19 ^{+ /+}The B cell.

Fig. 8 D shows with CD19 (thick line), CD20 (fine rule) or the painted B220 of pairing isotype contrast (CTL, dotted line) antibody (5 μ g/mL) ⁺The fluorescence intensity of cell, wherein specific by the concrete use of stream cell counting analysis isotype, PE bonded secondary antibody is carried out antibody staining.The result is presented at the fluorescence intensity that obtains in 4 experiments.The result is presented at from TG-1 ^+/-The spleen B220 of mouse ⁺The anti-hCD19 and the anti-mCD20 antibodies density of being correlated with on the B cell.No matter which antibody is used for each antibody, the anti-mCD20 antibodies of isotype density all is 10-64%, with anti-CD 19 antibodies density same high (Fig. 8 D).Though mCD20 expresses generally low than the hCD19 expression, at TG-I ^+/-In the mouse hCD19 expression levels still can with compare based on the hCD19 expression levels of human B cell (Figure 1B).Therefore, anti-CD 19 antibodies consumes the TG-2 that expresses hCD19 with relative low density effectively ^{+ /+}B cell (Figure 1B) is though pass through TG-1 ^{+ /+}With and TG-1 ^+/-The high-level CD19 of B cell expresses the relative different that has blured on the validity of IgG2a and IgG1 antibody.Though have direct retrocorrelation between the density that the quantity of B cell and hCD19 express in TG-I and TG-2 transgenic mice, the density of hCD19 is an important factor that promotes the B cell consumption.When with 250 μ g/ mouse administrations, the anti-CD 19 antibodies level be saturated (also referring to: the saturated level in Figure 12).Therefore, no matter the number of B cell, free anti-CD 19 antibodies level is excessive.

6.4 embodiment 3: organizing the B cell consumption is that FC γ R is dependent

Following test is used for determining whether to be depended on by the B cell consumption of anti-CD 19 antibodies the expression of FC γ R.By hCD19tg and some Fc γ R being lacked the method for the mouse interbreeding of expression, produced and expressed the mouse that hCD19 and some Fc γ R express.Such mouse is used to test measures anti-CD 19 antibodies for the approach by comprising that Fc γ R expresses, for example: ADCC consumes the ability of B cell.Therefore, the anti-CD 19 antibodies of Jian Dinging can utilize in the as above method described in 5.1 joints in these trials, be used to prepare chimeric, the people's or humanized anti-CD 19 antibodies.Such antibody can be used for the compositions and methods of the invention successively, treats the B cell malignancies in the human body.

The B cell consumption of process mediation after the anti-CD20 antibodies treatment that born immunity system relies on by Fc γ R.The mouse effector cell expresses four kinds of different Fc γ R types: IgG, high affinity Fc γ RI (CD64) and low affinity Fc γ RII (CD32), Fc γ RIII (CD6) and Fc γ RIV molecule.Fc γ RI, Fc γ RIII and Fc γ RIV are different oligopolymer mixtures, and its aglucon bonded α chain separately links to each other with common γ chain (FcR γ).For the triggering of Fc γ R assembling and Fc γ R effector function, the expression of FcR γ chain needs, and described effector function comprises the phagolysis by scavenger cell.Because FcR is γ ^-AMouse lacks high affinity Fc γ RI (CD64) and low affinity Fc γ RIII (CD 16) and Fc γ RIV molecule, so will express the FcR γ of hCD19 ^+/-Mouse be used for determining that Fc γ R is in the effect of organizing the B cell consumption after anti-CD 19 Antybody therapies.Fig. 9 A is presented at FcR γ ^+/-Or FcR γ ^-/-The consumption of seven days typical blood and spleen B cell after the treatment of the anti-CD19 of littermate or isotype control antibodies.Numeral indication B220 in specified path ⁺Lymphocytic per-cent.Fig. 9 B is presented at the 0th day FcR γ ^+/-Seven days blood and organize the consumption of B cell after the Antybody therapy of littermate.For blood, the value representative that after 0 of time, shows 1, the time data that obtain.The bar graph representative is at anti-CD19 (band of filling) of mouse (every group＞3 mouse) or the average B220 after isotype contrast (open band) Antybody therapy ⁺The B cell number.Indicated the significant difference between the mouse of anti-CD19 or the treatment of isotype control antibodies; ^*P＜0.05, ^*P＜0.01.The result who is present among Fig. 9 A and the 9B shows that the B cell consumption after the anti-CD 19 antibodies treatment is that FcR γ is dependent.When with the FcR γ of IgG2a antibody treatment with contrast ^-/-Littermate is compared, after the FMC63 Antybody therapy at FcR γ ^-/-The number of marrow, blood, spleen, lymphoglandula and peritoneal cavity B cell is not significant in the mouse changes.On the contrary, the anti-CD 19 antibodies treatment has consumed FcR γ ^+/-Most of B cell in the littermate.Therefore, the anti-CD 19 antibodies treatment mainly consumes blood and organizes the B cell by the approach that needs Fc γ RI and Fc γ RIII to express.

Fig. 9 C is presented at the hCD19TG-1 that monocyte consumes ^+/-Typical B cell number in the mouse.Treated mouse with clodronate disodium-liposome at the 2nd, 1 and 4 day, and gave FMC63 (n=9), isotype contrast (n=6) or CD20 (n=3) mAb (250 μ g) at the 0th day.With the mouse of PBS-liposome and FMC63 anti-CD 19 antibodies (n=3) treatment thing in contrast.With after Antybody therapy 7 days, the percentage of the appointment approach endolymph cell of indication recently showed typical blood and spleen B cell consumption.

Fig. 9 D show as in (C) behind Antybody therapy 7 days blood and organize the consumption of B cell.The average B220 of bar graph representative after the Antybody therapy of mouse (every group＞3 mouse) ⁺The B cell number.For blood, numerical value shows circulation B cell in the PBS treatment and the mouse of handling with FMC63 anti-CD 19 antibodies (closed triangle), and with the number of the monocyte consumption mouse of control antibodies (open circle), CD20 antibody (closed square) or FMC63 anti-CD 19 antibodies (closed circle) processing.Significant difference between mouse of having indicated and the average result of other groups in isotype contrast mAb treatment; ^*P＜0.05, ^*P＜0.01.

The B cell consumption that result among Fig. 9 is presented at after the anti-CD 19 antibodies treatment is that FcR γ and monocyte are dependent.Show mouse that scavenger cell lacks after FMC63, anti-CD20 (MB20-11) or the treatment of contrast anti-CD 19 antibodies 1 day by processing with liposomal encapsulated clodronate disodium, consume circulation B cell indistinctively, and the FMC63 Antybody therapy has been removed the circulation B cell (Fig. 9 C-D) in the mouse of the liposome-treated of loading with PBS.After 4-7 days, circulation B cell number is consumed significantly by FMC63 and anti-CD20 antibodies treatment, and the anti-CD 19 antibodies treatment has the effect that more attracts attention for the B cell number in the mouse of clodronate disodium treatment.Similarly, the treatment of anti-CD19 and anti-CD20 antibodies has reduced by 55% marrow B220 at the 7th day in the mouse of clodronate disodium treatment ⁺Cell number, and anti-CD 19 antibodies treatment has reduced by 88% marrow B220 exhausting in the 7th day in the mouse of PBS treatment ⁺Cell number.The anti-CD 19 antibodies treatment has reduced the number of 52% spleen B cell with respect to the littermate of control antibodies treatment in the mouse of clodronate disodium treatment at the 7th day, and the anti-CD20 antibodies minimally consumes the B cell in the mouse of PBS treatment, anti-CD 19 antibodies treatment minimizing the 89% spleen B cell number.The treatment of anti-CD19 and anti-CD20 antibodies is all at the number that reduced the lymphoglandula B cell of 48-53% behind the Antybody therapy in 7 days in the mouse of clodronate disodium treatment, and the anti-CD 19 antibodies treatment has reduced the number of 93% lymphoglandula B cell in the mouse of PBS treatment.In blood, spleen and lymphoglandula, the anti-CD 19 antibodies treatment is obviously than poor efficiency (p＜0.01) in the mouse of PBS treatment in the mouse of clodronate disodium treatment.These find that the hint scavenger cell is as body internal consumption CD19 ⁺And CD20 ⁺The main effector cell of B cell, and show that when monocyte number or function reduction the anti-CD 19 antibodies treatment may be more effective than the anti-CD20 antibodies treatment.

6.5 embodiment 4: anti-CD 19 antibodies inductive B cell consumption is persistent

For effectiveness and the time length of measuring the B cell consumption, to the single low dose of 250 μ g injections of hCD19TG mouse administration anti-CD 19 antibodies.Figure 10 A-1OC is illustrated in anti-CD 19 antibodies the treatment time length and the dose response of B cell consumption afterwards.Figure 10 A is presented at the 0th day FMC63 or isotype control antibodies treatment TG-I ^+/-After the mouse, blood B220 ⁺B cell and Thy-1 ⁺The number of T cell.Numerical value representative is from the mean value of every group of six mouse (± SEM) result.The result shows consumption circulation B cell in 13 weeks, and the B cell of blood carrying recovers gradually in ensuing 13 weeks.Thy-1 ⁺The T cell model is not that the result owing to anti-CD19 treatment changes.

Figure 10 B-10C is presented at 11,16 and 30 weeks after the Antybody therapy, is shown in as Figure 10 A and typically organizes the B cell consumption in the mouse.Numeral indication B220 in the path of formulating ⁺Lymphocytic per-cent.Result in Figure 10 B is presented at 11 weeks after the Antybody therapy, and marrow, blood, spleen, lymphoglandula and peritoneal cavity lack the B cell basically and (indicated the significant difference between sample mean; ^*P＜0.05, ^*P＜0.01).After first occurs at circulation B cell, reach normal range, get＞10 additional weeks in order to make circulation B cell number.Shown in Figure 10 C, in the 16th week behind Antybody therapy, the number of blood, spleen, LN and PLB cell has begun to recover, and BM B cell compartment is obviously not different with untreated contrast.To the 30th week, with the B cell can with level that normal control is compared on add all tissues again.

Figure 10 D shows the anti-CD 19 antibodies dosage that responds to blood, marrow and spleen B cell consumption.Treated mouse at the 0th day with anti-CD 19 antibodies, measured at the 7th day and organize the B cell model.The result shows that dosage for each antibody is by three the every group resulting values of mouse.The dosage of control antibodies is 250 μ g.Indicated the significant difference between sample mean; ^*P＜0.05, ^*P＜0.01.Low consumed the circulation B cell of remarkable quantity, and required 10 μ gHB 12b antibody to reduce the number (Figure 10 D) of circulation B cell significantly to the single FMC63 antibody dosage of 2 μ g/ mouse.5 times of the antibody dosages that need be higher than 10-50 μ g/ mouse to the remarkable consumption of the 7th day marrow and spleen B cell.Therefore, the CD19 Antybody therapy that carries out on low relatively dosage can consume for the major part circulation of important period and organize the B cell.

6.5.1 CD19 maintains the B cell surface after the anti-CD 19 antibodies administration

At HB12a, HB12b and FMC63 Antybody therapy (250 μ g) afterwards, determine by the expression of thinner cellular surface CD19 whether the internalization of CD19 influences the consumption of B cell in the body.

Figure 11 A-11C shows the TG-1 that uses HB12a (Figure 11 A), HB12b (Figure 11 B), FMC63 (Figure 11 C) or pairing isotype antibody (250 μ g) treatment in vivo ^+/-Cell surface CD19 expresses and the cleaning of B cell in the mouse.Time zero (before anti-CD19 administration) and behind antibody administration 1,4 and 24 hour, results spleen B cell, and with flowing the cell counting analysis by measuring CD19 (thick line) and contrast (fine rule) antibodies at the special secondary antibody processing cell of external use isotype.Also isolating B cell is added the secondary antibody that isotype is special with each CD19 antibody of saturation concentration, in the external expression of measuring total cell surface CD19 by stream cell counting analysis external.The result that each time point representative obtains from a mouse.Being present in result among Figure 11 A-11C shows in vivo after the antibodies, cell surface CD19 is not removed from cell surface, and show after Antybody therapy 24 hours, the high-caliber cell surface hCD19 that most spleen B cell expressing equates, the hypotype of B cell was expressed the hCD19 (Figure 11 C) of reduction level in 1 hour after the FMC63 Antybody therapy.Show also that in the result shown in Figure 11 A-11C the numerical value of CD19 on the B cell surface is steady state value, indication B cell is keeping mediating the ability of ADCC.

The result shows that after the anti-CD 19 antibodies administration CD19 shows surprisingly than the lower level internalization of expection.Especially, the result shows that CD19 maintains cell surface unexpectedly after the anti-CD 19 antibodies combination, and therefore, the B cell keeps that the ADCC activity can be arranged.These results partly show, why anti-CD 19 antibodies of the present invention and treatment plan are effective on treatment B cell malignancies.

Therefore the degree of Figure 12 A-12C proof B cell consumption and anti-hCD19 antibodies also stop the ability of other anti-hCD19 antibodies to hCD19.Result in Figure 12 A shows FMC63 TG-1 ^+/-The single administration of mouse (250 μ g) causes in the antibody administration obvious consumption of the B cell of blood and spleen within an hour.In this experiment, before the anti-CD 19 antibodies administration or at results blood of different thereafter time (1,4 or 24 hour) and splenocyte, and measure B cell frequency.Blood sample is identified the B cell with anti-Thy1.2 and anti-B220 dyeing in lower right quadrant.Splenocyte is identified the B cell with anti-IgM and anti-B220 antibody staining in specified path.The result that each time point representative obtains from a mouse.Unexpectedly, blood B cell is removed sooner than spleen B cell.

Show within an hour that at the B cell consumption described in Figure 12 A it is saturated that administered antibodies promptly makes hCD19 go up the available antibody binding site in administration.In order to confirm this observation, mouse is treated with FMC63 (hCD19 binding antibody) or isotype control antibodies.In the different thereafter time, blood and spleen B cell dyeing are identified at mCD19 with fluorescence dye bonded B4 antibody ⁺Or mCD20 ⁺Empty antibody binding site on the B cell surface.Pointed out the frequency of cell in the quadrant on the right of up and down.The result that each time point representative obtains from a mouse.The result shows that the FMC63 treatment causes the celliferous progressively consumption of hCD19 in process of the test, and wherein blood B cell consumes sooner than spleen.Those B cells of staying each time point place can dye by the expression of its mCD19 or mCD20 rather than by B4 and identify, illustrate that the FMC63 that gives combines with remaining B cell.These discoveries have confirmed FMC63 combination in vivo and the ability that consumes B.Result among Figure 12 C confirm administration within an hour HB12b Antybody therapy (250 μ g) also make the antibody binding site on the hCD19 saturated, and cause the consumption of the positive B cell of hCD19.Unexpectedly, HB12b antibody not exclusively suppresses the combination of B4 antibody, illustrate to be different from FMC63, and the epi-position on the HB12b identification hCD19, they are different with the identification by B4.Show that in the result shown in Figure 12 B-12C most anti-CD 19 antibodies suppresses the combination of other anti-CD 19 antibodies of great majority, show that most of anti-CD 19 antibodies are attached on CD19 proteic similar, identical or overlap or the epi-position.Optionally, these observations also may be owing to compare the relatively little size of CD19 extracellular domain with the size of antibody molecule.

6.6 embodiment 5: humoral immunization and autoimmunization have been eliminated in the anti-CD 19 antibodies treatment

Described in the present embodiment test can be used for determining immune response can be eliminated or reduce to anti-CD 19 antibodies whether.In these trials the anti-CD 19 antibodies of Jian Dinging can utilize as above the described method of 5.1 joints be used to prepare chimeric, the people's or humanized anti-CD 19 antibodies.Such antibody can be used for the compositions and methods of the invention successively, treats the B cell malignancies in the human body.

By giving hCD19tg ^+/-The single injection of mouse anti-CD 19 antibodies is determined at the effect of anti-CD 19 antibodies inductive B cell consumption on the serum antibody level.Figure 13 A shows that the CD19 Antybody therapy has reduced at TG-1 ^+/-The level of serum immune globulin in the mouse.At the 0th day single injection (closed circle) or two months big littermates of contrast (open circle) antibody (250 μ g) treatment with FMC63.Determine antibody horizontal by ELISA, adopt every group＞5 mean values shown in the mouse (± SEM).Difference between the mouse that the mAb of CD19 or contrast handles is tangible; ^*P＜0.05, ^*P＜0.01.The result was presented at after 1 to 2 week, and serum IgM, IgG2b, IgG3 and IgA antibody horizontal obviously reduce, and kept lowering at least 10 weeks (Figure 13 A).6 and 4 all IgG1 and IgG2a serum level are starkly lower than normally after processing.

Because hCD19TG ^+/-Mouse produces detectable autoantibody (people such as Sato, J.Immunol, 157:4371 (1996)) after 2 monthly ages, so can measure combining of serum autoantibody and ssDNA, dsDNA and histone.Figure 13 B is presented at after the anti-CD 19 antibodies treatment, the autoantibody level of anti-CD 19 antibodies treatment minimizing autoantibody anti-dsDNA, anti-ssDNA and anti-histone.The result is presented at the back anti-CD 19 antibodies treatment of 2 weeks and has obviously reduced serum IgM autoantibody level, and up to the generation (Figure 13 B) that stops isotype conversion IgG autoantibody 10 weeks.Therefore, the B cell consumption has reduced acute and secular antibody response in fact, and has reduced the type conversion of the immunne response of normal and morbidity.

By treating back 7 days, by with TNP-LPS or DNP-FicollB immunity hCD19TG at anti-CD 19 antibodies (FMC63) or control antibodies ^+/-Mouse (at the 0th day) is measured the influence of cell consumption for T cell independence Class1 (TI-1) and type 2 (TI-2) antibody response.Do not observe the special IgM of tangible haptens, IgG and IgA antibody response (Figure 14 A and 14B) in the mouse with the treatment of the anti-CD 19 antibodies of antigen immune.Also the mouse that utilized anti-CD 19 antibodies to treat in preceding 7 days in immunity is measured the antibody response (Figure 14 B) for T cell dependency (TD) Ag, DNP-KXH.Figure 14 C shows that the DNP-KLH mice immunized with the anti-CD 19 antibodies treatment has shown the immunity that is caused by body fluid that reduces.Before the 0th day initial immunity seven days with FMC63 (closed circle) or contrast (open circle) antibody (250 μ g) treatment littermate, day obtain serum formulating.For the DNP-KLH immunity, excite whole mouse at the 21st day DNP-KLH with 100 μ g.The AU value is to be used to average (± SEM) the ELISA ODU that obtains from the serum of every group of five mouse.Difference between the mouse of anti-CD19 or control antibodies treatment is tangible; ^*P＜0.05, ^*P＜0.01.The result shows that the littermate of control antibodies treatment produced elementary IgM antibody response in back 7 days in the DNP-KLH immunity, and produces secondary at the 21st day behind antigen stimulation and reply (Figure 14 C).But, in mouse, do not find that the special IgM of tangible haptens, IgG or IgA reply with antigen immune or the CD19mAb that excites again treatment.In order to determine the effect of B cell consumption on secondary antibody is replied, also after 14 days, use the DNP-KLH immune mouse, and treat (arrow) (Figure 14 D) with anti-CD 19 antibodies.By the 21st day, serum IgM, IgG and the anti-DNP antibody response of IgA dropped to the level that is lower than with the immune mouse antibody response of contrast mAb treatment in the mouse of CD19mAb treatment.But, the 21st day with DNP-KLH to exciting again that the mouse with contrast mAb treatment carries out, induced tangible secondary antibody to reply, and the mouse of CD19mAb treatment does not produce anti-DNP antibody after DNP-KLH excites again.Therefore, CD19mAb inductive B cell consumption has reduced elementary in fact and secondary antibody is replied, and has stoped type conversion during the immunne response that is caused by body fluid.

6.7 embodiment 6: the anti-CD 19 antibodies treatment combines with the anti-CD20 antibodies treatment

Test described herein can be used for determining other composition or combined treatment, and for example: anti-CD 19 antibodies combines whether have useful effect with chemotherapy, toxitherapy or radiotherapy, for example: one or more additives that increase the B cell consumption.By means known in the art, the result of tested combined therapy can be relevant with human body in animal model.

It is effective that anti-CD20 antibodies consumes on the B cell of people and mouse in vivo.So, assessed the benefit of using anti-CD19 (FMC63) and anti-CD20 (MB20-11) Antybody therapy simultaneously and determined whether it has increased the consumption of B cell.Use every kind of antibody of suboptimal 2 μ g dosage respectively, or the combination of two kinds of antibody of 1 μ g, or treat mouse with 2 μ g dosage of combination.Figure 15 is presented at the 0th day with contrast (250 μ g), FMC63 (CD 19,2 μ g), MB20-11 (CD20,2 μ g), FMC63 ⁺MB20 ^-11 (every kind 1 μ g) or FMC63 ⁺MB20 ^-11 (every kind 2 μ g) Antybody therapy TG-1 ^+/-The result of mouse.At time zero, measured the number of blood B cell in one hour and first, fourth and seven day.Value representative from the mean value of the group of three mouse (± SEM).Treatment was useful when result shown in Figure 15 showed anti-CD19 and anti-CD20 antibodies.The B cell consumption is intermediate or is similar to viewed consumption (Figure 15) in the mouse that the two kinds of antibody of the independent antibody 2 μ g with every kind are treated simultaneously in the mouse for the treatment of with two kinds of antibodies of 1 μ g.But, be used in two kinds of antibody under the 2 μ g and treat mouse simultaneously and cause than with the viewed obviously more B cell consumption of independent antibody.Therefore, the treatment of the anti-CD19 of associating and anti-CD20 antibodies has the beneficial effect that strengthens the B cell consumption.This may be owing to independently more treating the effectively accumulation of antibody molecule on the B cell surface.

6.8 embodiment 7: subcutaneous (S.C.) anti-CD 19 antibodies administration is that treatment is gone up effectively

Test described herein can be used for determining whether the subcutaneous administration approach of anti-CD 19 antibodies can consume the B cell effectively.By means known in the art, in animal model tested difference transport the result of the effectiveness of approach can be relevant with human body.

Because the anti-CD 19 antibodies of intravenously (i.v.) administration has consumed circulation effectively and organized the B cell, give anti-CD 19 antibodies and whether consume the B cell and reach equal degree so measure in subcutaneous (s.c.) or the abdomen (i.p.).FMC63 antibody with 250 μ g is treated wild-type mice with (i.p.) or intravenously (i.v.) administration in subcutaneous (s.c), the abdomen.The value representative was at the 7th day blood (every mL), marrow, spleen, lymphoglandula and the peritoneal cavity B220 by the stream determination of cell count ⁺The mean value of B cell number (± SEM).Pointed out the significant difference between the average result of every group of mouse; Compared with the control, ^*P＜0.05, ^*P＜0.01.Result in Figure 16 shows in subcutaneous (s.c.), the abdomen of CD19 antibody that (i.p.) or intravenously (i.v.) administration have consumed body-internal-circulation effectively and organized the B cell.Consuming most circulation and organizing B cell (Figure 16) in the mouse of (i.p.) or intravenously (i.v.) administration in anti-CD 19 antibody of 250 μ g dosage with subcutaneous (s.c.), abdomen.Unexpectedly, (i.p.) gives anti-CD 19 antibodies and consumes that belly B cell is not obvious to be better than intravenously (i.v.) administration in the abdomen.Therefore, when anti-CD 19 antibodies during with subcutaneous (s.c.) injection＜64mg, it can be used for consuming effectively circulation and organizes the B cell.Because anti-CD 19 antibodies is up to 10 μ g dosage still effectively (Figure 10 D), so even the antibody dosage of lower subcutaneous (s.c.) administration also is likely effective during intravenously (i.v.) administration.

6.9 embodiment 8: the growth of in-vivo tumour has been ended in the anti-CD 19 antibodies treatment

Burkitt's lymphoma, a kind of B cell malignancies in the human body, its characteristics are that the c-myc proto-oncogene translocates to the Ig gene promoter area, cause variant c-Myc to cross expression.Similarly, E μ-cMyc transgenosis (cMycTG) mouse, wherein the c-myc proto-oncogene is under the control of Ig heavy chain enhancer, produce the cell-derived malignant lymphoma of aggressive B in early days, about 90% mortality ratio was arranged to 20 ages in week, and at the survivor that median ages the time is arranged in about 12 weeks people such as (, people such as J.Exp.Med.167:353 (1988) and Adams, Nature 318:533 (1985)) Harris.Tumour from the c-MycTG mouse is not limited to the special B cell development stage, and mainly shows as the Ig gene rearrangement and the phenotypic characteristic (people such as Adams, Nature318:533 (1985)) of pre B cell or immature B cell.In order to determine in the body effectiveness, with hCD19TG-1 at the immunotherapy of CD19 ^{+ /+}Hybridize with the cMycTG mouse and to produce hCD19TG-1 ^+/-CMycTG ^{+ A}Mouse, it produces the cell-derived malignant lymphoma of aggressive B in early days.Separate the tumour cell that comes from a mouse, at amplification in vitro, and hCD19 on the performance phenotype ⁺With mouse CD19 ⁺CD20 ^-CD43 ^-IgM ⁺IgD ^-B220 ⁺Lymphoblastic characteristic, it has been represented at c-mycTG ^+/-The immature B cell tumour of the preceding B/ that produces in the mouse (people such as Harris, people such as J.Exp.Med.167:353 (1988) and Adams, Nature 318:533 (1985)).At the 0th day, will be from hCD19TG-l ^+/-C-mycTG ⁺The tumour cell (10 of mouse ⁵) intravenously (i.v.) is transplanted to 20 Rag ^+/-In the mouse.At the 1st and 7 day, with the mouse of selecting at random of FMC63 (fill circle) or contrast (open circle) antibody (250 μ g) treatment equal number.Figure 17 A is presented in cycle in 6 weeks circulating tumor cell by the stream determination of cell count, and (± SEM) number, Figure 17 B are presented at the per-cent of mouse survival rate in cycle in 7 weeks.Each value shows the per-cent of the survival mice of testing in every day.Result in Figure 17 shows that the anti-CD 19 antibodies treatment has suppressed hCD19 ⁺Malignant lymphoma development in vivo.With ten Rag of these tumor cell transplantations to two ^+/-In the mouse, cause two week back circulation mouse CD19 in ten recipients that select at random ⁺And B220 ⁺Lymphoblastic appearance, described recipient is with contrast mAb treatment, and dead to 3.5 weeks.By contrast, after tumour transplatation,, in whole ten recipients, stoped the appearance of circulating tumor cell up to 7 weeks with anti-CD 19 antibodies treatment ten mouse (the 1st and 7 day).The dead mouse of anti-CD 19 antibodies treatment during getting blood, but it does not demonstrate circulating tumor cell.Therefore, anti-CD 19 antibodies treatment may provide a kind of effective therapy that is used for the treatment of patient B cell system malignant tumour, especially those patients that do not express CD20 or express the tumour of CD20 on low-level.

Special embodiment described herein does not limit the scope of the invention.Certainly, by above description and accompanying drawing, the of the present invention different changes except that described will it will be apparent to those skilled in the art.Such change falls within the scope of accessory claim.

Quote various publications herein, its disclosed content is incorporated into herein as a reference in full with it.

Special embodiment described herein does not limit the scope of the invention.Certainly, by above description and accompanying drawing, the of the present invention different changes except that described will it will be apparent to those skilled in the art.Such change falls within the scope of accessory claim.

Quote various publications herein, its disclosed content is incorporated into herein as a reference in full with it.

International application no: PCT/US04105674

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From PCT/RO/134 (in January, 1981)

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＜223〉mouse anti human CD19 antibody B43 light chain V-kappa

<400>12

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Arg Ser

<210>13

<211>107

<212>PRT

＜213〉mouse

<220>

＜223〉mouse anti human CD19 antibody 63FMC light chain V-kappa

<400>13

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

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

100 105

<210>14

<211>115

<212>PRT

＜213〉mouse

<220>

＜223〉mouse anti human CD19 antibody 4G7 light chain V-kappa

<400>14

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

1 5 10 15

Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu Asn Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His

85 90 95

Leu Glu Tyr Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

Arg Ala Asp

115

<210>15

<211>494

<212>DNA

＜213〉mouse

<220>

<221>CDS

<222>(57)...(494)

＜223〉mouse anti human CD19 antibody HB12a light chain

<400>15

catggactga aggagtagaa aactgatcac tctcctatgt ttatttcctc aaaatg atg 59

Met

1

agt cct gcc cag ttc ctg ttt ctg tta gtg ctc tgg att cag gaa acc 107

Ser Pro Ala Gln Phe Leu Phe Leu Leu Val Leu Trp Ile Gln Glu Thr

5 10 15

aac ggt gat gtt ggg atg acc cag act cca ctc act ttg tcg gtc acc 155

Asn Gly Asp Val Gly Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr

20 25 30

att gga caa cca gcc tct ttc tct tgc aag tca agt cag agc ctc tta 203

Ile Gly Gln Pro Ala Ser Phe Ser Cys Lys Ser Ser Gln Ser Leu Leu

35 40 45

tat agt aat gga aaa acc tat ttg aat tgg tta tta cag agg cca ggc 251

Tyr Ser Asn Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly

50 55 60 65

cag tct cca aag cgc cta atc cat ctg gtg tct aaa ctg gac tct gga 299

Gln Ser Pro Lys Arg Leu Ile His Leu Val Ser Lys Leu Asp Ser Gly

70 75 80

gtc cct gac agg ttc act ggc agt gga tca gga aca gat ttt aca ctg 347

Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

85 90 95

aaa atc ggc aga gtg gag gct gag gat ttg gga gtt tat tac tgc gtg 395

Lys Ile Gly Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Val

100 105 110

caa ggt aca cat ttt ccg tac acg ttc gga ggg ggg acc aaa cta gaa 443

Gln Gly Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu

115 120 125

ata aaa cgg gct gat gct gca cca act gta tcc atc ttc cca cca tcc 491

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

130 135 140 145

agt 494

Ser

<210>16

<211>146

<212>PRT

＜213〉mouse

<220>

＜223〉Yu Ce mouse anti human CD19 antibody HB12a light chain

<400>16

Met Ser Pro Ala Gln Phe Leu Phe Leu Leu Val Leu Trp Ile Gln Glu

1 5 10 15

Thr Asn Gly Asp Val Gly Met Thr Gln Thr Pro Leu Thr Leu Ser Val

20 25 30

Thr Ile Gly Gln Pro Ala Ser Phe Ser Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Tyr Ser Asn Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro

50 55 60

Gly Gln Ser Pro Lys Arg Leu Ile His Leu Val Ser Lys Leu Asp Ser

65 70 75 80

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

85 90 95

Leu Lys Ile Gly Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys

100 105 110

Val Gln Gly Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu

115 120 125

Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro

130 135 140

Ser Ser

145

<210>17

<211>485

<212>DNA

＜213〉mouse

<220>

<221>CDS

<222>(48)...(485)

＜223〉mouse anti human CD19 antibody HB12b light chain

<400>17

catggactga aggagtagaa aagcattctc tcttccagtt ctcagag atg gag aaa 56

Met Glu Lys

1

gac aca ctc ctg cta tgg gtc ctg ctt ctc tgg gtt cca ggt tcc aca 104

Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro Gly Ser Thr

5 10 15

ggt gac att gtg ctg acg cag tct cca acc tct ttg gct gtg tct cta 152

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

20 25 30 35

ggg cag agg gcc acc atc tcc tgc aga gcc agc gaa agt gtt gat act 200

Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Thr

40 45 50

ttt ggc att agt ttt atg aac tgg ttc caa cag aaa cca gga cag cca 248

Phe Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro

55 60 65

ccc aaa ctc ctc atc cat gct gca tcc aat caa gga tcc ggg gtc cct 296

Pro Lys Leu Leu Ile His Ala Ala Ser Asn Gln Gly Ser Gly Val Pro

70 75 80

gcc agg ttt agt ggt agt ggg tct ggg acg gac ttc agc ctc aac atc 344

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

85 90 95

cat cct atg gag gag gat gat agt gca atg tat ttc tgt cag caa agt 392

His Pro Met Glu Glu Asp Asp Ser Ala Met Tyr Phe Cys Gln Gln Ser

100 105 110 115

aag gag gtt cca ttc acg ttc ggc tcg ggg aca aag ttg gaa ata aaa 440

Lys Glu Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

120 125 130

cgg gct gat gct gca cca act gta tcc atc ttc cca cca tcc agt 485

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser

135 140 145

<210>18

<211>146

<212>PRT

＜213〉mouse

<220>

＜223〉mouse anti human CD19 antibody HB12b light chain

<400>18

Met Glu Lys Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

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

20 25 30

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

35 40 45

Val Asp Thr Phe Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro

50 55 60

Gly Gln Pro Pro Lys Leu Leu Ile His Ala Ala Ser Asn Gln Gly Ser

65 70 75 80

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser

85 90 95

Leu Asn Ile His Pro Met Glu Glu Asp Asp Ser Ala Met Tyr Phe Cys

100 105 110

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

115 120 125

Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro

130 135 140

Ser Ser

145

<210>19

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉mix the justice 5 ' VH primer (MsVHE) of having dwelt

<400>19

gggaattcga ggtgcagctg caggagtctg g 31

<210>20

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉be complementary to the antisense primer (primer C1) of C coding region

<400>20

gagttccagg tcactgtcac tggctcaggg a 31

<210>21

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉constant region specific antisense 3 ' primer

<400>21

gactgaggca cctccagatg ttaactg 27

Claims (63)

1. pharmaceutical composition, it comprises in the pharmaceutically acceptable carrier, IgG1 or IgG3 people's isotype, mono-clonal, people or Humanized anti-cd 19 antibodies.

2. pharmaceutical composition, its comprise in the pharmaceutically acceptable carrier, the treatment significant quantity, IgG1 or IgG3 people's isotype, the chimeric anti-CD 19 antibodies of mono-clonal.

3. the pharmaceutical composition of claim 2, wherein the treatment significant quantity of IgG1 or the chimeric anti-CD 19 antibodies of IgG3 people's isotype mono-clonal is less than the about 1mg of every kg of patient body weight.

4. the pharmaceutical composition of claim 2, wherein the treatment significant quantity of IgG1 or the chimeric anti-CD 19 antibodies of IgG3 people's isotype mono-clonal is greater than the about 2mg of every kg of patient body weight.

5. claim 1 or 2 pharmaceutical composition, wherein anti-CD 19 antibody are IgG1 people's isotype antibodies.

6. claim 1 or 2 pharmaceutical composition, wherein anti-CD 19 antibody are IgG3 people's isotype antibodies.

7. pharmaceutical composition, its comprise in the pharmaceutically acceptable carrier, the treatment significant quantity, Mediated Human antibody dependent cellular mediation cytotoxicity (ADCC), monoclonal human or Humanized anti-cd 19 antibodies.

8. pharmaceutical composition, its comprise cytotoxicity (ADCC) in the pharmaceutically acceptable carrier, the mediation of Mediated Human antibody dependent cellular, the chimeric anti-CD 19 antibodies of mono-clonal.

9. claim 7 or 8 composition, wherein anti-CD 19 antibody are people's isotypes of IgG1, IgG2, IgG3 or IgG4.

10. claim 1,2,7 or 8 pharmaceutical composition, wherein anti-CD 19 antibodies has at least 4 to 7 days half life.

11. claim 1,2,7 or 8 pharmaceutical composition, wherein to comprise dosage be 1500mg/m to composition ²Or anti-CD 19 antibodies still less.

12. the pharmaceutical composition of claim 11, wherein to comprise dosage be 375mg/m to composition ²Or anti-CD 19 antibodies still less.

13. the pharmaceutical composition of claim 11, wherein to comprise dosage be 1.5mg/m to composition ²Or anti-CD 19 antibodies still less.

14. the pharmaceutical composition of claim 13, wherein composition comprises dosage 0.5 μ g/m ²Or anti-CD 19 antibodies still less.

15. claim 1,2,7 or 8 pharmaceutical composition, wherein anti-CD 19 antibodies is with detectable mode mark.

16. claim 1,2,7 or 8 pharmaceutical composition, wherein anti-CD 19 antibodies is naked antibody.

17. claim 1,2,7 or 8 pharmaceutical composition, wherein anti-CD 19 antibodies combines with therapeutic compound.

18. claim 1,2,7 or 8 pharmaceutical composition, wherein anti-CD 19 antibodies combines with cytotoxin reagent.

19. claim 1,2,7 or 8 pharmaceutical composition, wherein anti-CD 19 antibodies combines with diagnostic reagent.

20. claim 1,2,7 or 8 pharmaceutical composition, wherein anti-CD 19 antibodies is a dual specific.

21. the pharmaceutical composition of claim 20, wherein the anti-CD 19 antibodies of dual specific has for the specificity in conjunction with the effector cell.

22. the pharmaceutical composition of claim 7, wherein the ADCC function of anti-CD 19 antibodies is to determine by the target cell dissolving power of effector cell's mediation by measuring anti-CD 19 antibodies in vivo.

23. claim 1,2,7 or 8 pharmaceutical composition, wherein anti-CD 19 antibodies comprises the heavy chain CDR3 district of antibody HB 12a or HB12b, or has the heavy chain CDR3 district of at least 25% amino acid sequence identity with the heavy chain CDR3 district of HB12a or HB12b antibody.

24. the pharmaceutical composition of claim 23, wherein anti-CD 19 antibodies comprises heavy chain CDR1, CDR2 and the CDR3 district of HB12a or HB12b antibody, or heavy chain CDR1, the CDR2 and the CDR3 district that have at least 25% amino acid sequence identity with heavy chain CDR1, CDR2 and the CDR3 district of HB12a or HB12b antibody.

25. a method for the treatment of B cell malignancies in the human body, it comprises uses the amount that is enough to consume circulation B cell, IgG1 or IgG3 people's isotype, monoclonal human or Humanized anti-cd 19 antibodies to required patient.

26. a method for the treatment of B cell malignancies in the human body, its comprise to required patient use the cytotoxicity (ADCC) amount, the mediation of Mediated Human antibody dependent cellular that is enough to consume circulation B cell, monoclonal human or Humanized anti-cd 19 antibodies.

27. a method for the treatment of B cell malignancies in the human patients, it comprises required patient's administering therapeutic significant quantity, IgG1 or IgG3 people's isotype, monoclonal human or Humanized anti-cd 19 antibodies.

28. a method for the treatment of B cell malignancies in the human patients, its comprise to cytotoxicity (ADCC) required patient's administering therapeutic effective scheme, Mediated Human antibody dependent cellular mediation, mono-clonal, people or Humanized anti-cd 19 antibodies.

29. the method for claim 26 or 28, wherein anti-CD 19 antibodies has IgG1, IgG2, IgG3 or IgG4 people's isotype.

30. claim 25,26,27 or 28 method, wherein treatment B cell malignancies earlier before anti-CD 19 antibody administrations.

31. the method for claim 30, wherein the treatment of malignant tumour is chemotherapy, radiotherapy, toxitherapy, prodrug activation enzyme treatment, antibody therapy, monocyte or scavenger cell intensive cure, immune modulating treatment, statin treatment, calicheamycin treatment, surgical operation therapy or its any combination.

32. claim 25,26,27 or 28 method are wherein used the therapy for treating B cell malignancies except the anti-CD 19 antibodies treatment after the anti-CD 19 antibodies administration.

33. the method for claim 32, wherein the treatment of malignant tumour is chemotherapy, radiotherapy, toxitherapy, prodrug activation enzyme treatment, antibody therapy, monocyte or scavenger cell intensive cure, immune modulating treatment, statin treatment, calicheamycin treatment, surgical operation therapy or its arbitrary combination.

34. a method for the treatment of the early stage disease that is caused by the B cell malignancies in the human patients, it comprises the monoclonal anti-CD 19 antibodies to the cytotoxicity (ADCC) of the Mediated Human antibody dependent cellular mediation of required patient's administering therapeutic effective scheme.

35. method for the treatment of B cell malignancies in the human patients, it comprises the monoclonal anti-CD 19 antibodies to the cytotoxicity (ADCC) of the Mediated Human antibody dependent cellular mediation of required experimenter's administering therapeutic effective scheme, and wherein said experimenter did not accept the treatment of malignant tumour in the past.

36. the method for claim 35, it further comprises and gives the treatment of human patients except anti-CD 19 Antybody therapies subsequently.

37. the method for claim 35 or 36, wherein treatment be chemotherapy, radiotherapy, based on the treatment of toxin, based on radiochemical treatment or surgical operation therapy.

38. method for the treatment of B cell malignancies in the human patients, it comprises monoclonal anti-CD 19 antibody to the cytotoxicity (ADCC) of the Mediated Human antibody dependent cellular mediation of required patient's administering therapeutic effective scheme, and wherein the B cell malignancies is the CD19 male.

39. method for the treatment of B cell malignancies in the human patients, it comprises the monoclonal anti-CD 19 antibodies to the cytotoxicity (ADCC) of the Mediated Human antibody dependent cellular mediation of required patient's administering therapeutic effective scheme, and wherein human patients has the monocyte number of one of every at least dL.

40. claim 27,28,34,35,38 or 39 method, wherein therapy comprises the administration of antibody as the single therapy agent.

41. claim 27,28,34,35,38 or 39 method, wherein therapy comprises antibody and another kind of therapeutical agent co-administered.

42. claim 27,28,34,35,38 or 39 method, wherein therapy comprises antibody co-administered with the reagent that reduces the toxic agent side effect.

43. claim 27,28,34,35,38 or 39 method, wherein the scheme of administration has consumed circulation B cell.

44. claim 27,28,34,35,38 or 39 method, wherein therapy comprises with the antibody of the amount of enough consumption circulation B cells and uses repeatedly.

45. claim 25,26,27,28,34,35,38 or 39 method, wherein anti-CD 19 antibodies has at least 4 to 7 days half life.

46. claim 34,35,38 or 39 method, wherein anti-CD 19 antibodies is human IgG1's isotype antibody.

47. claim 34,35,38 or 39 method, wherein anti-CD 19 antibodies is human IgG 3 isotype antibodies.

48. claim 34,35,38 or 39 method, wherein anti-CD 19 antibodies is human IgG2's isotype antibody.

49. claim 34,35,38 or 39 method, wherein anti-CD 19 antibodies is human IgG 4 isotype antibodies.

50. claim 34,35,38 or 39 method, wherein anti-CD 19 antibodies is people or humanized antibody.

51. claim 25,26,27,28,34,35,38 or 39 method, wherein the B cell malignancies is a B cell subsets Fei Huoqijinshi malignant lymphoma (NHL), and it comprises low etc./folliculus NHL, small lymphocyte (SL) NHL, medium/folliculus NHL, medium diffusivity NHL, high immunoblast NHL, high lymphocytoblast NHL, high little non-cleaved cell NHL and huge disease NHL; Burkitt's lymphoma; Multiple myeloma; Preceding B acute lymphoblastic leukemia and other derive from the malignant tumour of early stage B cell precursor; The common acute Lymphocytic leukemia; Lymphocytic leukemia; Hairy cell leukemia; Non-acute lymphoblastic leukemia; The Fahrenheit macroglobulinemia; And PL; Light chain disease; Plasmoma; The osteosclerosis myelomatosis; Plasma cell leukemia; The MG (MGUS) that feature is not clear; Smouldering property multiple myeloma (SMM); Slow property multiple myeloma (IMM) or hodgkin's malignant lymphoma.

52. claim 27,28,34,35,38 or 39 method, wherein therapy further comprises and uses the compound that strengthens monocyte or macrophage function.

53. the method for claim 52, wherein the people is an impaired immune response.

54. claim 25,26,27,28,34,35,38 or 39 method are wherein used anti-CD 19 antibodies by approach in parenteral, the abdomen or intramuscular injection.

55. claim 25,26,27,28,34,35,38 or 39 method are wherein used anti-CD 19 antibodies by intravenously or subcutaneous approach.

56. the method for claim 55, wherein by subcutaneous approach with 37.5mg/m ²Or dosage is still less used anti-CD 19 antibody.

57. the method for claim 56, wherein by subcutaneous approach with 1.5mg/m ²Or dosage is still less used anti-CD 19 antibodies.

58. each method in the claim 25,26,27,28,34,35,38 or 39, the B cell that wherein circulates reaches at least 75% consumption.

59. the method for claim 58, the consumption of wherein said circulation B cell is observed at least 7 days time.

60. the method for claim 58, the consumption of wherein said circulation B cell is observed at least 30 days time.

61. the method for claim 58, the consumption of wherein said circulation B cell is observed at least 6 months time.

62. each method in the claim 27,28,34,35,38 or 39, wherein therapy comprises human body is used once or above anti-CD 19 antibodies.

63. each method in the claim 27,28,34,35,38 or 39, wherein therapy further comprises and gives anti-CD20 antibodies, anti-CD22 antibody, anti-CD 52 antibody or its arbitrary combination.

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