CN101379090A

CN101379090A - Antibodies to OX-2/CD200 and uses thereof

Info

Publication number: CN101379090A
Application number: CNA200780005048XA
Authority: CN
Inventors: K·S·鲍迪什; A·克雷茨-罗梅尔; M·S·法斯; J·P·斯普林霍恩; 邬大洋
Original assignee: Alexion Pharmaceuticals Inc
Current assignee: Alexion Pharmaceuticals Inc
Priority date: 2006-01-12
Filing date: 2007-01-11
Publication date: 2009-03-04

Abstract

This application provides methods and compositions for modulating and/or depleting CD200 positive cells.

Description

Antibody of anti-OX-2/CD200 and uses thereof

Related application

The application requires the U.S. Provisional Application submitted on January 12nd, 2006 number 60/758,426, the U.S. Provisional Application of submitting on January 12nd, 2006 number 60/759, the U.S. Provisional Application of submitting on May 18th, 085 and 2006 number 60/801,991 right of priority are with complete being incorporated herein by reference of these applications.

Technical field

The disclosure relates to OX-2/CD200 (being called CD200 herein) antagonist and exhaust or eliminated the method for the cell of expressing CD200 in the experimenter who suffers from cancer or autoimmune disease.The therapy of treatment cancer provides the combination of two kinds of mechanism.More particularly, the disclosure relates to the use therapy for treatment of cancer, described therapy: (1) disturbs the interaction between CD200 and its acceptor to suppress with blocking immunity, thereby promotes the elimination of cancer cells; And/or (2) (a) cytotoxicity by antibody dependent cellular cytotoxicity or complement-mediated or use by (b) and to comprise that the fusion molecule target of CD200 target certain portions decides the direct kill cancer cell of cell.The disclosure also relates to the method by the therapy for treating autoimmune disease, and described therapy increases the antibody dependent cellular cytotoxicity of the positive immunocyte of CD200 and/or the cytotoxicity of complement-mediated.

Background

Multiple mechanism works in the replying of morbid state (comprising cancer) at health.For example, CD4 ⁺T helper cell plays a crucial role in the effective immunne response at multiple malignant tumour by stimulating factor is provided for the effector cell.Think that cytotoxic T cell is to eliminate the most effective cell of cancer cells, and t helper cell passes through secretion Th1 cytokine such as IL-2 and IFN-γ and trigger cell toxicity T cell.In multiple malignant tumour, shown that t helper cell compares the phenotype with change with the cell of finding in healthy individual.A kind of feature of outstanding change is that the Th1 cytokine that reduces produces and is partial to produce the Th2 cytokine and (for example sees that Kiani waits the people, Haematologica88:754-761 (2003); Maggio waits the people, Ann Oncol 13 Suppl 1:52-56 (2002); Ito waits the people, Cancer 85:2359-2367 (1999); Podhorecka waits the people, Leuk Res 26:657-660 (2002); Tatsumi waits the people, J Exp Med 196:619-628 (2002); Agarwal waits the people, Immunol Invest 32:17-30 (2003); Smyth waits the people, Ann Surg Oncol 10:455-462 (2003); Contasta waits the people, Cancer Biother Radiopharm 18:549-557 (2003); Lauerova waits the people, Neoplasma 49:159-166 (2002) .).Shown that making cytokine be partial to the antitumous effect that Th1 spectrum can strengthen the T cell (sees Winter, wait the people, Immunology108:409-419 (2003); Inagawa waits the people, Anticancer Res 18:3957-3964 (1998) .).

The cytokine-expressing that tumour cell can drive t helper cell comprises the surface molecular of secrete cytokines such as IL-10 and TGF-β and expression and immune cell interaction from the potential mechanism of Th1 deflection Th2.CD200 is relevant with immunosuppression, and CD200 is a kind of molecule of expressing on the surface of dendritic cell, and the molecule of itself and immunoglobulin gene family has high homology (people such as Gorczynski, Transplantation 65:1106-1114 (1998)).Shown that the cell of for example expressing CD200 can suppress the stimulation of Th1 cytokine production.

Although immunocyte can help to attack and eliminate cancer cells, in some cases, as in autoimmune disease, transformation reactions and tissue or organ graft repulsion, immunity system can cause disease.In order to suppress deleterious immune response in this type of situation, can use immunosuppressor to the patient, as reflunomide and cytokine antagonist.Yet these general immunosuppression can cause undesirable side effect, comprise cytotoxicity and the resistance reduction to infecting.Thereby, need be alternative, the autoimmune method of treatment that may be more special.

More verified immunomodulatory therapies comprise that antibody therapy is successful in the treatment of some cancer and autoimmune disease.Yet, need extra antibody therapy to treat cancer and autoimmune disease clinically.In addition, relevant humanization or other the chimeric people/mouse monoclonal antibodies of also needing.In known research, the patient who is applied mouse-anti-TNF (tumour necrosis factor) monoclonal antibody has produced anti-mouse antibody reaction (Exley A.R. waits the people, Lancet 335:1275-1277 (1990)) to the antibody of being used.(so-called human anti-mouse antibody (HAMA) replys (people Q J Nucl Med Mol Imaging 2004 such as Mirick in the immune response to treatment plan of the type; 48:251-7), reduced the validity of treatment and even make that treatment is invalid fully.Shown that humanization or chimeric people/mouse monoclonal antibody significantly reduce the treatment validity that HAMA replied and increased Antybody therapy.For example see people such as LoBuglio, P.N.A.S.86:4220-4224 (June 1989).In addition, wherein particular functionality is enhanced or the antibody that weakens can have useful application in clinical.

General introduction

The disclosure relates to material and the method that is used to regulate the CD200 function.The material of regulating the CD200 function comprises the material of regulating the active of CD200 and/or its acceptor (CD200R) and/or expressing.In some embodiments, described material suppresses function or the activity of CD200.Thereby in some respects, described material is as the antagonist of CD200.Some antagonists can be incorporated into CD200 and suppress or destroy the interaction of CD200 and its acceptor.Other antagonists can be incorporated into CD200 but not blocking CD200:CD200R interacts.Thereby the CD200 antagonist comprises and can regulate any material of CD200 effect by comprising or not comprise the interactional mechanism of blocking-up CD200:CD200R.The CD200 antagonist includes but not limited to polypeptide, small molecules, organometallic compound, oligonucleotide construct, RNAi construct, aptamers, spiegelmers, antisense nucleic acid, locked nucleic acid (LNA) inhibitor, peptide nucleic acid(PNA) (PNA) inhibitor, immunomodulator, antibody, Fab, prodrug and/or peptide simulated compound.

In some embodiments, described antagonist is anti-CD200 antibody.The antibody that relates to as this paper comprises Fab, Fab, Fv, scFv, Fab ' and F (ab ') ₂, mono-clonal and polyclonal antibody, through engineering approaches antibody (antibody that comprises antibody, humanized antibody, fully human antibodies and the artificial selection of chimeric antibody, single-chain antibody, CDR grafting) and synthetic or semisynthetic antibody.

In some respects, the disclosure relate to chimeric, humanized, the people's and remove anti-CD200 antibody and its Fab of immunity.In some embodiments, antibody of the present disclosure comprises heavy chain, and this heavy chain comprises and is selected from the aminoacid sequence that SEQ ID NOS:7,9,11 and 20 aminoacid sequence or its fragment have at least 90% identity.Comprise comprise to SEQ ID NOS:7,9,11 and 20 in the antibody of the same or similar aminoacid sequence of the aminoacid sequence that provides or its fragment (including but not limited to fragment) about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% corresponding to the sequence that does not have leader sequence.Described antibody can additionally comprise light chain, this light chain comprise to be selected from SEQ ID NOS:24,26,28 with 32 aminoacid sequence or its fragment at least about 90% same or similar aminoacid sequence.Similarly, aforementioned aminoacid sequence can with the aminoacid sequence that provides among the SEQ ID NOS:24,26,28 and 32, comprise that its fragment (including but not limited to the fragment corresponding to the sequence that does not have leader sequence) about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% is same or similar.

In one embodiment, the disclosure relates to the anti-CD200 antibody that comprises heavy chain and light chain, heavy chain comprises aminoacid sequence with SEQ ID NO:7 at least about 90% same aminoacid sequence, light chain comprise with SEQ ID NO:24 at least about 90% same aminoacid sequence.Also comprise anti-CD200 antibody, its comprise to SEQ ID NOS:7 and 24 in the same or similar aminoacid sequence of one or more aminoacid sequences of providing or its fragment about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.Fragment includes, but are not limited to the sequence that does not have leader sequence corresponding to the sequence that provides in SEQ IDNOS:7 and 24.Therefore, the disclosure relates to anti-CD200 antibody, it is included in the aminoacid sequence of the nucleic acid sequence encoding of hybridizing with the nucleotide sequence (comprising its fragment and its complementary sequence) of SEQ ID NO:6 under the stringent condition, and is also contained in the aminoacid sequence of the nucleic acid sequence encoding of hybridizing with the nucleotide sequence (comprising its fragment and its complementary sequence) of SEQ ID NO:23 under the stringent condition.Also comprise anti-CD200 antibody, its comprise to SEQ IDNO:6 in the nucleotide sequence (comprising its fragment and its complementary sequence) that provides at least about the aminoacid sequence of 80% homology or similar nucleic acid sequence encoding, also comprise to SEQ ID NO:23 in the nucleotide sequence (comprising its fragment and its complementary sequence) that provides at least about the aminoacid sequence of 80% homology or similar nucleic acid sequence encoding.The invention still further relates to anti-CD200 antibody, its comprise to SEQ ID NO:6 or 23 in the nucleotide sequence (comprising its fragment and its complementary sequence) that provides at least about the aminoacid sequence of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology or similar nucleic acid sequence encoding.

In another embodiment, the disclosure relates to anti-CD200 antibody, its comprise contain with the aminoacid sequence of SEQ IDNO:9 at least about 90% same aminoacid sequence and also comprise with SEQ IDNO:26 at least about 90% same aminoacid sequence.Also comprise anti-CD200 antibody, it comprises and SEQ ID NOS:9 and 26 or the same or similar aminoacid sequence of one or more aminoacid sequences of providing of its fragment about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.Fragment includes but not limited to corresponding to the sequence that provides in SEQ ID NOS:9 and 26, does not have the sequence of leader sequence.Therefore, the present invention relates to anti-CD200 antibody, it is included in the aminoacid sequence of the nucleic acid sequence encoding of hybridizing with the nucleotide sequence (comprising its fragment and its complementary sequence) of SEQ ID NO:8 under the stringent condition, and is also contained in the aminoacid sequence of the nucleic acid sequence encoding of hybridizing with the nucleotide sequence (comprising its fragment and its complementary sequence) of SEQID NO:25 under the stringent condition.Also comprise anti-CD200 antibody, its comprise to SEQ ID NO:8 in the nucleotide sequence (comprising its fragment and its complementary sequence) that provides at least about the aminoacid sequence of 80% homology or similar nucleic acid sequence encoding, also comprise to SEQ ID NO:25 in the nucleotide sequence (comprising its fragment and its complementary sequence) that provides at least about the aminoacid sequence of 80% homology or similar nucleic acid sequence encoding.The invention still further relates to anti-CD200 antibody, its comprise to SEQ ID NO:8 or 25 in the nucleotide sequence (comprising its fragment and its complementary sequence) that provides at least about the aminoacid sequence of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology or similar nucleic acid sequence encoding.

In another embodiment, the disclosure relates to anti-CD200 antibody, its comprise with the aminoacid sequence of SEQ ID NO:11 at least about 90% same aminoacid sequence and also comprise with SEQ ID NO:26 at least about 90% same aminoacid sequence.Also comprise anti-CD200 antibody, it comprises and SEQID NOS:11 and 26 or the same or similar aminoacid sequence of one or more aminoacid sequences of providing of its fragment about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.Fragment includes but not limited to corresponding to the sequence that provides in SEQ ID NOS:11 and 26, does not have the sequence of leader sequence.Therefore, the present invention relates to anti-CD200 antibody, it is included in the aminoacid sequence of the nucleic acid sequence encoding of hybridizing with the nucleotide sequence (comprising its fragment and its complementary sequence) of SEQ ID NO:10 under the stringent condition, and is also contained in the aminoacid sequence of the nucleic acid sequence encoding of hybridizing with the nucleotide sequence (comprising its fragment and its complementary sequence) of SEQ ID NO:25 under the stringent condition.Also comprise anti-CD200 antibody, its comprise to SEQ ID NO:10 in the nucleotide sequence (comprising its fragment and its complementary sequence) that provides at least about the aminoacid sequence of 80% homology or similar nucleic acid sequence encoding, also comprise to SEQ ID NO:25 in the nucleotide sequence (comprising its fragment and its complementary sequence) that provides at least about the aminoacid sequence of 80% homology or similar nucleic acid sequence encoding.The invention still further relates to anti-CD200 antibody, its comprise to SEQ ID NO:10 or 25 in the aminoacid sequence of about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of nucleotide sequence (comprising its fragment and its complementary sequence) that provides or similar nucleic acid sequence encoding.

In extra embodiment, the disclosure relates to anti-CD200 antibody, its comprise with the aminoacid sequence of SEQ IDNO:11 at least about 90% same aminoacid sequence and also comprise with SEQ IDNO:28 at least about 90% same aminoacid sequence.Also comprise anti-CD200 antibody, it comprises and SEQ ID NOS:11 and 28 or the same or similar aminoacid sequence of one or more aminoacid sequences of providing of its fragment about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.Fragment includes but not limited to corresponding to the sequence that provides in SEQ ID NOS:11 and 28, does not have the sequence of leader sequence.Therefore, the present invention relates to anti-CD200 antibody, it is included in the aminoacid sequence of the nucleic acid sequence encoding of hybridizing with the nucleotide sequence (comprising its fragment and its complementary sequence) of SEQ ID NO:10 under the stringent condition, and is also contained in the aminoacid sequence of the nucleic acid sequence encoding of hybridizing with the nucleotide sequence (comprising its fragment and its complementary sequence) of SEQIDNO:27 under the stringent condition.Also comprise anti-CD200 antibody, its comprise to SEQ ID NO:10 in the nucleotide sequence (comprising its fragment and its complementary sequence) that provides at least about the aminoacid sequence of 80% homology or similar nucleic acid sequence encoding, also comprise to SEQ ID NO:27 in the nucleotide sequence (comprising its fragment and its complementary sequence) that provides at least about the aminoacid sequence of 80% homology or similar nucleic acid sequence encoding.The invention still further relates to anti-CD200 antibody, its comprise to SEQ ID NO:10 or 27 in the aminoacid sequence of about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of nucleotide sequence (comprising its fragment and its complementary sequence) that provides or similar nucleic acid sequence encoding.

In a further embodiment, the disclosure relates to anti-CD200 antibody, its comprise with the aminoacid sequence of SEQ IDNO:20 at least about 90% same aminoacid sequence and also comprise with SEQ IDNO:32 at least about 90% same aminoacid sequence.Also comprise anti-CD200 antibody, it comprises and SEQ ID NOS:20 and 32 or the same or similar aminoacid sequence of one or more aminoacid sequences of providing of its fragment about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.Fragment includes but not limited to corresponding to the sequence that provides in SEQ ID NOS:20 and 32, does not have the sequence of leader sequence.Therefore, the disclosure relates to anti-CD200 antibody, it is included in the aminoacid sequence of the nucleic acid sequence encoding of hybridizing with the nucleotide sequence (comprising its fragment and its complementary sequence) of SEQ ID NO:19 under the stringent condition, and is also contained in the aminoacid sequence of the nucleic acid sequence encoding of hybridizing with the nucleotide sequence (comprising its fragment and its complementary sequence) of SEQID NO:31 under the stringent condition.Also comprise anti-CD200 antibody, its comprise to SEQ ID NO:19 in the nucleotide sequence (comprising its fragment and its complementary sequence) that provides at least about the aminoacid sequence of 80% homology or similar nucleic acid sequence encoding, also comprise to SEQ ID NO:31 in the nucleotide sequence (comprising its fragment and its complementary sequence) that provides at least about the aminoacid sequence of 80% homology or similar nucleic acid sequence encoding.Therefore the present invention comprises anti-CD200 antibody, its comprise to SEQ ID NO:19 or 31 in the aminoacid sequence of about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of nucleotide sequence (comprising its fragment and its complementary sequence) that provides or similar nucleic acid sequence encoding.

The anti-CD200 antibody that provides in the disclosure comprises having antibody and Fab change or that do not have the effect subfunction.Comprise the constant region that comprises change or the antibody in Fc district, it has effector function increase or that reduce.The disclosure also relates to owing to the binding affinity that increases or reduce has antibody change or that do not have the effect subfunction, and binding affinity described increase or that reduce can cause owing to the change of variable region.The effector function that changes for example comprises, the ability that strengthens or weaken, increase or the cytotoxicity (ADCC) of the dependence antibody that reduces, and/or increase or the cytotoxicity (CDC) of the dependence complement that reduces in conjunction with one or more Fc acceptors (FcR) or effector cell.Variation antibody includes but not limited to such antibody, and wherein contain one or more aminoacid insertion, disappearance and/or substitute in constant region or Fc district.In extra embodiment, these variation antibody comprise constant region, wherein CH1 and hinge area from human IgG2 and CH2 and CH3 district from human IgG 4.Also comprise antibody, wherein constant or Fc district demonstrates the glycosylation of change.Aforementioned antibody and Fab (comprising single-chain antibody) comprise mouse, chimeric, humanized, complete the people's or go the immunity; Comprise the antibody that comprises IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgA, IgD or IgE framework.In addition, described antibody (comprising its fragment and variant) can be closure or misclosure antibody or its fragment.

In some respects, the disclosure provides and has demonstrated the anti-CD200 antibody that reduces or do not have the effect subfunction.Have Fc or constant region that the antibody that reduces or do not have the effect subfunction can comprise variation or change, as have the constant region of one or more amino acid replacements, insertion and/or disappearance, or have the constant region that one or more glycosylations change.The variation constant region for example comprises, such zone, and wherein one or more amino acid are substituted by L-Ala, Ala-Ala sudden change as described herein, perhaps wherein one or more carbohydrate groups are changed, add or remove.Can realize the change of carbohydrate group number and/or position by produce described antibody in particular cell types, in described cell type, posttranslational modification will reduce, lack or increase.In one embodiment, remove the effector function of anti-CD200 antibody by exchange IgG1 constant domain and IgG2/4 fusion structure territory.It is contemplated that the additive method of removing effector function, as suddenly change known and the interactional site of FcR or the insertion of peptide in hinge area, thereby remove the FcR required critical sites that interacts.

Aspect more of the present disclosure and in the method, have anti-CD200 antibody change or that do not have the effect subfunction and comprise anti-CD200 antibody with one or more amino acid replacements, insertion and/or disappearance.In some embodiments, the anti-CD200 antibody of this type of variation demonstrate reduce or do not have an effect subfunction.In some embodiments, the constant region of (described variation antibody) variation and native sequences is constant or Fc district and/or have at least about 70% homology with the constant of parental antibody or Fc district or its fragment; In other embodiments, the constant or Fc district of variation has with it at least about 80% homology or similarity; In other embodiments, have with it, in extra embodiment, have with it at least about 95% homology or similarity at least about 90% homology or similarity.In specific embodiments, variation antibody comprises the G2/G4 construct.Therefore, the disclosure relates to the constant or Fc district with the anti-CD200 antibody that reduces or do not have the effect subfunction, and wherein said constant region comprises heavy chain, and this heavy chain comprises and is selected from SEQ ID NOS:13,15,18,22 and its segmental aminoacid sequence.The disclosure also relates to the variation constant region of anti-CD200 antibody, and wherein this antibody comprises and is selected from SEQ IDNOS:13,15,18,22, with its segmental aminoacid sequence at least about 90% same or similar aminoacid sequence.Also comprise antibody in the disclosure, this antibody comprise to SEQ ID NOS:13,15,18,22 in the same or similar aminoacid sequence of the amino acid that provides and its fragment about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.Fragment includes, but not limited to not have the sequence of leader sequence.In addition, in some embodiments, has the constant region of anti-CD200 antibody that reduces or do not have the effect subfunction and comprise the G2/G4 construct by being selected from SEQ ID NOS:12,14,16,17 and 21 or the nucleic acid encoding of its fragment and its complementary sequence.In some embodiments, has the anti-CD200 antibody that reduces or do not have the effect subfunction by comprising and the nucleic acid encoding of the sequence that is selected from SEQ IDNOS:12,14,16,17 and 21 (comprising its fragment and complementary sequence) at least about 80% homology or similar nucleotide sequence.In other embodiments, the anti-CD200 antibody of variation is by the nucleic acid sequence encoding that comprises to about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology of the nucleotide sequence that is selected from SEQ ID NOS:12,14,16,17 and 21 (comprising its fragment and complementary sequence) or similar sequence.In other embodiments, the nucleic acid of the anti-CD200 antibody of coding variation is included under the stringent condition nucleotide sequence with the nucleic acid array hybridizing that is selected from SEQID NOS:12,14,16,17 and 21 (comprising its fragment and complementary sequence).Comprise Fab and closure and misclosure antibody or its fragment.

In one embodiment, the anti-CD200 antibody that the present invention relates to make a variation, its comprise with the aminoacid sequence of SEQ IDNO:13 at least about 90% same aminoacid sequence and also comprise aminoacid sequence with SEQ IDNO:28 at least about 90% same aminoacid sequence.Also comprise anti-CD200 antibody, its comprise with SEQ ID NOS:13 and 28 in the same one or more aminoacid sequences of the aminoacid sequence that provides or its fragment about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.Fragment includes, but not limited to the sequence that does not have leader sequence corresponding to the sequence that provides in SEQ ID NOS:13 and 28.Therefore, the disclosure relates to the anti-CD200 antibody of variation, it is included in the aminoacid sequence of the nucleic acid sequence encoding of hybridizing with the nucleotide sequence (comprising its fragment and its complementary sequence) of SEQ ID NO:12 under the stringent condition, and is also contained in the aminoacid sequence of the nucleic acid sequence encoding of hybridizing with the nucleotide sequence (comprising its fragment and its complementary sequence) of SEQ ID NO:27 under the stringent condition.Also comprise anti-CD200 antibody, its comprise with SEQID NO:12 in the nucleotide sequence (comprise its fragment and its complementary sequence) that provides at least about the aminoacid sequence of 80% homologous nucleic acid sequence encoding, also comprise with SEQ ID NO:27 in the nucleotide sequence (comprising its fragment and its complementary sequence) that provides at least about the aminoacid sequence of 80% homologous nucleic acid sequence encoding.Therefore comprise anti-CD200 antibody, its comprise with SEQ ID NO:12 or 27 in the nucleotide sequence (comprising its fragment and its complementary sequence) that provides at least about the aminoacid sequence of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous nucleic acid sequence encoding.

In another embodiment, the disclosure relates to the anti-CD200 antibody of variation, its comprise with the aminoacid sequence of SEQID NO:15 at least about 90% same aminoacid sequence and also comprise aminoacid sequence with SEQID NO:24 at least about 90% same aminoacid sequence.Also comprise anti-CD200 antibody, its comprise with SEQ ID NOS:15 and 24 in the same aminoacid sequence of one or more aminoacid sequences of providing or its fragment about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.Fragment includes, but not limited to the sequence that does not have leader sequence (for example, the fragment that begins at amino acid 20 or 21 places of SEQ ID NO:15) corresponding to the sequence that provides in SEQ ID NOS:15 and 24.Therefore, the disclosure relates to the anti-CD200 antibody of variation, it is included in the aminoacid sequence of the nucleic acid sequence encoding of hybridizing with the nucleotide sequence (comprising its fragment and its complementary sequence) of SEQ ID NO:14 under the stringent condition, and is also contained in the aminoacid sequence of the nucleic acid sequence encoding of hybridizing with the nucleotide sequence (comprising its fragment and its complementary sequence) of SEQ ID NO:23 under the stringent condition.Also comprise anti-CD200 antibody, its comprise with SEQ ID NO:14 in the nucleotide sequence (comprise its fragment and its complementary sequence) that provides at least about the aminoacid sequence of 80% homologous nucleic acid sequence encoding, also comprise with SEQ ID NO:23 in the nucleotide sequence (comprising its fragment and its complementary sequence) that provides at least about the aminoacid sequence of 80% homologous nucleic acid sequence encoding.Therefore comprise anti-CD200 antibody, its comprise with SEQ ID NO:14 or 23 in the aminoacid sequence of about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, the 99% or 100% homologous nucleic acid sequence encoding of nucleotide sequence (comprising its fragment and its complementary sequence) that provides.

In extra embodiment, the anti-CD200 antibody that the present invention relates to make a variation, its comprise with the aminoacid sequence of SEQID NO:13 at least about 90% same aminoacid sequence and also comprise the aminoacid sequence same with the aminoacid sequence at least 90% of SEQID NO:28.Also comprise anti-CD200 antibody, its comprise with SEQ ID NOS:13 and 28 in the same one or more aminoacid sequences of the aminoacid sequence that provides or its fragment about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.Fragment includes, but not limited to the sequence that does not have leader sequence corresponding to the sequence that provides in SEQ ID NOS:13 and 28.Therefore, the disclosure relates to the anti-CD200 antibody of variation, it is included in the aminoacid sequence of the nucleic acid sequence encoding of hybridizing with the nucleotide sequence (comprising its fragment and its complementary sequence) of SEQ ID NO:16 under the stringent condition, and is also contained in the aminoacid sequence of the nucleic acid sequence encoding of hybridizing with the nucleotide sequence (comprising its fragment and its complementary sequence) of SEQ ID NO:27 under the stringent condition.Also comprise anti-CD200 antibody, its comprise with SEQID NO:16 in the nucleotide sequence (comprise its fragment and its complementary sequence) that provides at least about the aminoacid sequence of 80% homologous nucleic acid sequence encoding, also comprise with SEQ ID NO:27 in the nucleotide sequence (comprising its fragment and its complementary sequence) that provides at least about the aminoacid sequence of 80% homologous nucleic acid sequence encoding.Therefore comprise anti-CD200 antibody, its comprise with SEQ ID NO:16 or 27 in the aminoacid sequence of about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, the 99% or 100% homologous nucleic acid sequence encoding of nucleotide sequence (comprising its fragment and its complementary sequence) that provides.

In a further embodiment, the disclosure relates to the anti-CD200 antibody of variation, its comprise with the aminoacid sequence of SEQIDNO:18 at least about 90% same aminoacid sequence and also comprise aminoacid sequence with SEQID NO:30 at least about 90% same aminoacid sequence.Also comprise anti-CD200 antibody, its comprise with SEQ ID NOS:18 and 30 in the same aminoacid sequence of the aminoacid sequence that provides or its fragment about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.Therefore, the disclosure relates to the anti-CD200 antibody of variation, it is included in the aminoacid sequence of the nucleic acid sequence encoding of hybridizing with the nucleotide sequence (comprising its fragment and its complementary sequence) of SEQ ID NO:17 under the stringent condition, and is also contained in the aminoacid sequence of the nucleic acid sequence encoding of hybridizing with the nucleotide sequence (comprising its fragment and its complementary sequence) of SEQ ID NO:29 under the stringent condition.Also comprise anti-CD200 antibody, its comprise with SEQ ID NO:17 in the nucleotide sequence (comprise its fragment and its complementary sequence) that provides at least about the aminoacid sequence of 80% homologous nucleic acid sequence encoding, also comprise with SEQ ID NO:29 in the nucleotide sequence (comprising its fragment and its complementary sequence) that provides at least about the aminoacid sequence of 80% homologous nucleic acid sequence encoding.Therefore comprise anti-CD200 antibody, its comprise with SEQ ID NO:17 or 29 in the aminoacid sequence of about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, the 99% or 100% homologous nucleic acid sequence encoding of nucleotide sequence (comprising its fragment and its complementary sequence) that provides.

In another embodiment, the anti-CD200 antibody that the present invention relates to make a variation, its comprise with the aminoacid sequence of SEQ ID NO:22 at least about 90% same aminoacid sequence and also comprise aminoacid sequence with SEQ ID NO:34 at least about 90% same aminoacid sequence.Also comprise anti-CD200 antibody, its comprise with SEQ ID NOS:22 and 34 in the same aminoacid sequence of the aminoacid sequence that provides or its fragment about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.Therefore, the disclosure relates to the anti-CD200 antibody of variation, it is included in the aminoacid sequence of the nucleic acid sequence encoding of hybridizing with the nucleotide sequence (comprising its fragment and its complementary sequence) of SEQ ID NO:21 under the stringent condition, and is also contained in the aminoacid sequence of the nucleic acid sequence encoding of hybridizing with the nucleotide sequence (comprising its fragment and its complementary sequence) of SEQ IDNO:33 under the stringent condition.Also comprise anti-CD200 antibody, its comprise with SEQ ID NO:21 in the nucleotide sequence (comprise its fragment and its complementary sequence) that provides at least about the aminoacid sequence of 80% homologous nucleic acid sequence encoding, also comprise with SEQ ID NO:33 in the nucleotide sequence (comprising its fragment and its complementary sequence) that provides at least about the aminoacid sequence of 80% homologous nucleic acid sequence encoding.Therefore comprise anti-CD200 antibody, its comprise with SEQ ID NO:21 and 33 in the aminoacid sequence of about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, the 99% or 100% homologous nucleic acid sequence encoding of nucleotide sequence (comprising its fragment and its complementary sequence) that provides.

Anti-CD200 antibody with effector function of change can demonstrate the effector function of increase.The effector function that increases include but not limited to the increase of one or more Fc acceptors combination, cause that the ability of ADCC increases, and/or cause that the ability of CDC increases.Anti-CD200 antibody with effector function of increase also comprises variation Fc or constant region as described herein.Aforementioned anti-CD200 antibody with effector function of change can also be closure or misclosure antibody.For example, but the anti-CD200 antibody with effector function of increase can be incorporated into CD200 can not block the CD200:CD200R combination.When with the cell of effector function (for example, ADCC or CDC) targeted expression CD200, can use this antibody-like.As previously mentioned, antibody described herein, comprise aforementioned anti-CD200 antibody with effector function of change comprise mouse, chimeric, humanized, complete the people's and remove the antibody of immunity, all are their closure and misclosure form and its fragment.

In some respects, the disclosure provides the method and composition that is used to regulate or exhaust the CD200 positive cell.By regulating or exhaust the CD200 positive cell to experimenter's administration of anti-cd 20 0 antagonist.Described antagonist can be decided the CD200 positive cell and/or can destroy CD200:CD200R to interact by target because of effector function.In some embodiments, described antagonist is anti-CD200 antibody.Described anti-CD200 antibody can be antibody described herein, comprises its any fragment and variant.Comprise the antibody and the Fab of effector function, as have anti-CD200 antibody reduction or that do not have the effect subfunction with change.Also comprise mouse, chimeric, humanized, complete the people's and go the immunity antibody and Fab, comprise single-chain antibody.Aforementioned antibody can be misclosure or blocking antibody and comprise the antibody that comprises IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgA, IgD or IgE framework.

The CD200 positive cell relates to cancer and some autoimmune disease of some type.Therefore, the CD200 positive cell includes but not limited to, immunocyte (as, B cell and T cell) and cancer cells (as the cancer cells of ovarian cancer, skin carcinoma, lung cancer, kidney, mammary cancer, prostate cancer, neuroblastoma, lymphoma, myelomatosis, leukemia, thyroid carcinoma and plasmocyte cancer).Also comprise from any tissue of neural crest cell or the cancer cells of organ.Thereby, need to regulate or the experimenter that exhausts the CD200 positive cell can be the patient who suffers from cancer or autoimmune disease, perhaps accepted or expected the patient who accepts organ transplantation.

On the one hand, the disclosure provides the method and composition of treatment autoimmune disease.Include but not limited to rheumatoid arthritis, inflammatory bowel (comprising ulcerative colitis and crohn), systemic lupus erythematous, multiple sclerosis, Hashimoto thyroiditis, pernicious anemia, Addison disease, type i diabetes, dermatomyositis, Sjogren syndrome, lupus erythematosus, myasthenia gravis, reiter syndrome, Graves' disease, psoriatic and autoimmune hemolytic by the treatable autoimmune disease of method and composition provided herein.In some embodiments, the patient who suffers from autoimmune disease is given the antagonist of CD200, and in some embodiments, antagonist is anti-CD200 antibody.Anti-CD200 antibody can comprise variation constant region as described herein.Therefore, anti-CD200 antibody can demonstrate the effector function of change, as the effector function that increases.Described antibody for example can demonstrate the combination to the increase of one or more Fc acceptors.In addition, described antibody can cause enhanced ADCC and/or CDC.Described antibody can also be closure or misclosure antibody or its fragment and can be mouse, chimeric, humanized, complete the people's or go antibody or its fragment of immunity.

Can use the cancer of disclosed method treatment to include but not limited to melanoma, ovarian cancer, kidney, neuroblastoma, lung cancer, mammary cancer, prostate cancer, lymphoma, myelomatosis, leukemia and plasmocyte cancer.Also comprise from the cancer of neural crest cell and any cancer of expression CD200.In some embodiments, the disclosure provides the treatment hematologic malignancies, as leukemia, comprises the method for chronic lymphocytic leukemia.

In the embodiment that is particularly useful, comprise according to cancer therapy of the present disclosure that (1) uses and disturb between CD200 and its acceptor interactional anti-CD200 antibody or antagonist to suppress, thereby promote the removing of cancer cells with blocking immunity; And/or (2) use comprise CD200 target certain portions fusion molecule with direct kill cancer cell.Alternatively, antibody is by the direct kill cancer cell of cytotoxicity complement-mediated and/or that rely on antibody.In multiple embodiments, the effector function of anti-CD200 antibody is changed.In a specific embodiments, the constant region that anti-CD200 antibody contains variation or changes, its effector function is weakened or is eliminated; The method that (1) and (2) was described above this antibody for example can be used for.

In some embodiments, the disclosure relates to fusion molecule, and wherein anti-CD200 antibody or Fab are connected to second kind of molecule.Described fusion molecule for example can comprise, small molecules, polypeptide, peptide mimics, irregular (heteroclitic) peptide, chemotherapeutic, immunomodulator, targeting moiety, perhaps nucleic acid construct (as antisense constructs, RNAi or gene targeting construct).The disclosure also comprises the Fab at CD200, and wherein said fragment is fused to or is connected to polypeptide, protein domain, serum protein, albumin, PEG (polyoxyethylene glycol), maybe will increase any other molecule of half life in the described segmental body.Described Fab for example comprises, Fab, Fv, single-chain fragment or scFv, Fab ' and F (ab ') ₂

The disclosure also relates to the method for the CD200 expression status that uses the cell or tissue sample that anti-CD200 TPPA obtains from the patient.This method includes but not limited to, the immunohistochemical staining of tissue sample and from the flow cytometry of patient's CD200 staining cell.The patient can be the patient who for example suffers from cancer.

According to method and composition as herein described, the disclosure also relates to the method for the treatment of transplanting or allotransplantation patient.Can transplant or the allotransplantation step before or after the step patient used anti-CD200 antibody of the present disclosure or other CD200 antagonists so that reduce or eliminates the positive immunocyte of CD200, described CD200 positive immunocyte can reduce the acceptance of patient to transplanted organ or tissue.In specific embodiments, the transplant patient had the anti-CD200 antibody of the effector function of increase.

The method of the combination treatment that is used to comprise anti-CD200 therapy is provided in other embodiments.For example, the patient who accepts to comprise first kind of therapy of CD200 antagonist (for example, anti-CD200 antibody as herein described) also can be given second kind of therapy.The CD200 antagonist can give simultaneously with second kind of therapy.Alternatively, the CD200 antagonist can give before or after second kind of therapy.Second kind of therapy includes but not limited to chemotherapeutic, radiotherapy, vaccine, microbiotic and antiviral agent and immunomodulatory therapy.

In another embodiment of the present disclosure, provide the method for monitor therapy therapeutic advance.This method relates to be used therapy (for example, immunomodulatory therapy, chemotherapy therapy, or the like) and at least twice and measures among the experimenter CD200 level to determine the validity of this therapy.Other methods of measuring therapy validity include but not limited to, detect in cancer cells, total lymphocyte counting, spleen, liver and/or lymphoglandula size, regulatory T cells number, the cell or the serum cytokines spectrum, perhaps as the secretion of the cytokine of T that measures by ELISPOT or B cell, ELISPOT is a kind of mensuration system that allows based on each cell detection cytokine or other excretory molecules.

According to composition that provides in the present embodiment and method, the disclosure also relates to the arbitrary pharmaceutical composition that comprises anti-CD200 antibody.Comprise chimeric, humanized, people and remove the anti-CD200 antibody and the Fab of immunity, comprise single-chain antibody.Also comprise mouse, chimeric, humanized, the people of the effector function with change as described herein and remove variation anti-CD 20 0 antibody of immunity.Aforementioned antibody and variation antibody can be closure or misclosure antibody or Fab.

In some embodiments, can use the patient of anti-CD200 therapy or expection to accept to comprise treatment or step that the patient of the therapy of CD200 antagonist therapy (comprising for example anti-CD200 antibody) can be screened accepts before some or screen current medical condition.For example, in one embodiment, can screen gestation in advance and agree contraception female patient, because CD200 plays an important role in the protection at miscarriage.Therefore, the patient who accepts described therapy can agree to implement one or more contraceptive devices.Described patient can be with being intended to begin in the preceding designated duration of described therapy and/or one or more contraceptive devices of use in the time length of described therapy.In some embodiments, female patient accepts to be exposed to about fetus the consulting of the danger of this anti-CD200 therapy.In extra embodiment, can expect that the patient signs the informed consent table before this treatment.In other respects, the consulting patient about the doctor of anti-CD200 therapy can need this type of patient before using anti-CD200 therapy, to use to practise contraception apparatus or preparation (for example see, US6,908,432 with relevant patent, its content is incorporated herein by reference).Similarly, in other embodiments, can screen the patient to accept the radiotherapeutic patient of brain operation and/or brain before identifying; To not advise anti-CD200 therapy for this type of patient.

The accompanying drawing summary

Fig. 1 provides the nucleotide sequence (SEQID NO:1) of the primer C7mhHF that is used to produce the G2/G4 construct.

Fig. 2 provides the nucleotide sequence (SEQ ID NO:2) of the primer Rev Age Pri that is used to produce the G2/G4 construct.

Fig. 3 provides the nucleotide sequence (SEQID NO:3) of the primer C2aB7 rev that is used to produce the G2/G4 construct.

Fig. 4 provides the nucleotide sequence (SEQ ID NO:4) of the lacpri that is used to produce the G2/G4 construct.

Fig. 5 provides the nucleotide sequence (SEQ ID NO:5) of LeadVHpAX.

Fig. 6 A-F has described the heavy chain of antibody chC2aB7-hG1 and the aminoacid sequence and the nucleotide sequence (SEQ ID NOS:6,7,23,24,37 and 38) of light chain.Fig. 6 C has shown SEQ ID NO:37 (nucleotide sequence) and SEQ ID NO:7 (aminoacid sequence).As the SEQID NO:7 that shows in the synoptic diagram be successive but with comprising that the corresponding nucleotide sequences of intron describes.Fig. 6 F has shown SEQ ID NO:38 (nucleotide sequence) and SEQ ID NO:24 (aminoacid sequence).

Fig. 7 A-F has described the heavy chain of antibody hB7V4V1-hG1 and the aminoacid sequence and the nucleotide sequence (SEQ ID NOS:8,9,25,26,39 and 40) of light chain.Fig. 7 C has shown SEQ ID NO:39 (nucleotide sequence) and SEQ ID NO:9 (aminoacid sequence).Fig. 7 F has shown SEQ ID NO:40 (nucleotide sequence) and SEQ ID NO:26 (aminoacid sequence).

Fig. 8 A-F has described the heavy chain of antibody hB7V3V1-hG1 and the aminoacid sequence and the nucleotide sequence (SEQ ID NOS:10,11,25,26,40 and 41) of light chain.Fig. 8 C has shown SEQ ID NO:41 (nucleotide sequence) and SEQ ID NO:11 (aminoacid sequence).Fig. 8 F has shown SEQ ID NO:41 (nucleotide sequence) and SEQ ID NO:26 (aminoacid sequence).

Fig. 9 A-F has described the heavy chain of antibody hB7V3V2-hG1 and the aminoacid sequence and the nucleotide sequence (SEQ ID NOS:10,11,27,28,41 and 42) of light chain.Fig. 9 C has shown SEQ ID NO:41 (nucleotide sequence) and SEQ ID NO:11 (aminoacid sequence).Fig. 9 F has shown SEQ ID NO:42 (nucleotide sequence) and SEQ ID NO:28 (aminoacid sequence).

Figure 10 A-F has described the heavy chain of antibody hB7V3V2-hG2G4 and the aminoacid sequence and the nucleotide sequence (SEQ IDNOS:12,13,27,28,42 and 43) of light chain.Figure 10 C has shown SEQID NO:43 (nucleotide sequence) and SEQ ID NO:13 (aminoacid sequence).As the SEQ ID NO:13 that shows in the synoptic diagram be successive but with comprising that the corresponding nucleotide sequence of intron describes.Figure 10 F has shown SEQ ID NO:42 (nucleotide sequence) and SEQ ID NO:28 (aminoacid sequence).

Figure 11 A-F has described the heavy chain of antibody chC2aB7-hG2G4 and the aminoacid sequence and the nucleotide sequence (SEQ ID NOS:14,15,23,24,44,45,46 and 47) of light chain.Figure 11 C has shown SEQ ID NO:44 (nucleotide sequence) and SEQ ID NO:45 (aminoacid sequence).SEQID NO:45 is corresponding to the amino acid/11-337 of SEQ ID NO:15.Show as synoptic diagram, SEQ IDNO:45 be successive but with comprising that the corresponding nucleotide sequence of intron describes.Figure 11 F has shown SEQ ID NO:46 (nucleotide sequence) and SEQ ID NO:47 (aminoacid sequence).

Figure 12 A-F has described the heavy chain of antibody hB7V3V2-cG2G4 and the aminoacid sequence and the nucleotide sequence (SEQ ID NOS:13,16,27,28,48 and 49) of light chain.Figure 12 C has shown SEQID NO:48 (nucleotide sequence) and SEQ ID NO:13 (aminoacid sequence).Figure 12 F has shown SEQ ID NO:49 (nucleotide sequence) and SEQ ID NO:28 (aminoacid sequence).

Figure 13 A-D has described the heavy chain of antibody chC7-hG2G4 and the aminoacid sequence and the nucleotide sequence (SEQ ID NOS:17,18,29 and 30) of light chain.

Figure 14 A-F has described the heavy chain of antibody D1B5-hG1 and the aminoacid sequence and the nucleotide sequence (SEQ ID NOS:19,20,31,32,50 and 51) of light chain.Figure 14 C has shown SEQ ID NO:50 (nucleotide sequence) and SEQ ID NO:20 (aminoacid sequence).The SEQ IDNO:20 that shows as synoptic diagram be successive but with comprising that the corresponding nucleotide sequence of intron describes.Figure 14 F has shown SEQ ID NO:51 (nucleotide sequence) and SEQ ID NO:32 (aminoacid sequence).

Figure 15 A-D has described the heavy chain of antibody G2G4 63L1D and the aminoacid sequence and the nucleotide sequence (SEQ ID NOS:21,22,33 and 34) of light chain.

Figure 16 provides the nucleotide sequence (SEQ IDNO:35) of the forward primer that is used to clone CD200 cDNA.

Figure 17 provides the nucleotide sequence (SEQ IDNO:36) of the reverse primer that is used to clone CD200cDNA.

Figure 18 has shown the effect of using humanization CD200 antibody in the RAJI-CD200/PBL model.Humanized anti-CD 20 0 antibody causes the inhibition of tumor growth.

Figure 19 has illustrated the effect of using the humanization CD200 antibody that is with or without effector function in the Namalwa_CD200 animal model.There is not the antibody of effector function in suppressing tumor growth, to demonstrate effect.

Figure 20 is a form, has shown with normal specimens to compare the expression level of CD200 in chronic lymphocytic leukemia (CLL) patient sample.

Figure 21 has described the level relatively that detected CD200 expresses in cancerous cell line.

Figure 22 has shown with respect to detected expression level in human peripheral lymphocyte (PBL), the antigenic expression level of CD200 in the human ovarian cancer sample.

Figure 23 has shown with respect to detected expression level in PBL, the antigenic expression level of CD200 in human melanoma patient sample.

Figure 24 has shown the immunohistochemical staining of the CD200 of melanoma patient sample.

Figure 25 has illustrated anti-CD200 antibody in the aborning effect of cytokine.CD200 antibody do not exist and in the presence of measure the blended cell colony measure in IL-2 generation level.The antibody that uses is the inosculating antibody CD200 antibody that does not have effector function.

Figure 26 has shown the effect of using the anti-CD200 antibody that is with or without effector function in the Namalwa/PBL model, and in described model, tumour is not expressed CD200.

Figure 27 has shown the flow cytometry that CD200 expresses on the activated T cell.The CD3+ cell is activated with mOKT3, results, wash and use pointed to people CD25, CD200, CD5, CD4 and CD8 special put together antibody staining.Harvested cell is also analyzed immunofluorescence with CellQuest software on the FacsCaliber flow cytometer.

Figure 28 illustrates the effect of anti-CD200 antibody to the ADCC of activated T cell.The CD3+ human T-cell was dyeed 72 hours with 10 μ g/mL immobilized (dull and stereotyped bag quilt) mOKT3.Then with the activated T cell with chromate treating with as target and with as effector cell's purifying from body CD56+ (NK) cell incubation.Cell is with the effector cell of 25:1 (A) or 10:1 (B): target cell than can mediate effector function (V3V2-G1) or lack through transformation effector function (V3V2-G2G4) Humanized anti-CD 20 0 antibody existence or not down at 37 ℃ of incubations 4 hours altogether down.The special cracking of data represented per-cent.Anti-CD200 antibody has increased the ADCC of activated T cell, and does not have the anti-CD200 antibody of effector function can not induce ADCC.

Figure 29 is a form, has shown the expression level of CD200 on plasmocyte.

DESCRIPTION OF THE PREFERRED

The I.CD200 antagonist

CD200 is the I type transmembrane glycoprotein of the high conservative of expressing on the various kinds of cell type, described cell type (for example comprises immune cell, T cell, B cell and dendritic cell (people such as Barclay, TRENDS Immunol.2002:23)) and some cancer cells, as described herein.Its acceptor CD200R (the people J.Immunol.2003 (171) such as Wright: people such as 3034-3046 and Wright, Immunity2000 (13): 233-242) that interacts on the subpopulation of this protein and medullary cell and T cell; CD200:CD200R interacts the immunomodulatory signal delivery is suppressed to cell and induction of immunity, comprises the relevant immunotolerance of apoptosis (people 2004 Blood (103) such as Rosenblum: 2691-2698).Thereby, disturb the function or the active material of CD200 and/or its acceptor can suppress or reverse the interactional immunosuppressive effect of CD200:CD200R.In addition, the material of specific combination CD200 (interacting but can suppress or not suppress CD200:CD200R) can cause the downstream events that reverses or eliminate the effect of CD200.

In some respects, the disclosure relates to the CD200 antagonist.As used herein, the term antagonist comprises any material of activity, function and/or the expression that can suppress CD200 or its acceptor.Example (for example includes but not limited to polypeptide, antibody, small molecules, aptamers, spiegelmers, locked nucleic acid (LNA) inhibitor, peptide nucleic acid(PNA) (PNA) inhibitor, nucleic acid construct, gene targeting construct, antisense constructs, RNA disturb (RNAi) construct, or the like) and peptide mimics.In some embodiments, antagonist destroys the interaction of CD200 and CD200R.In other embodiments, the CD200 antagonist cell that can reduce the immunosuppressive effect of CD200 or can target express CD200 surely is used for exhausting or removing.

In some respects, the CD200 antagonist is a polypeptide.Being used for polypeptide of the present disclosure can make up with different technologies well known by persons skilled in the art.In one embodiment, obtain polypeptide by chemosynthesis.In other embodiments, polypeptide is from a fragment of one or more antibody or the antibody of several fragment structure.In other embodiments, described polypeptide is an anti-CD200 antibody as described herein.

Thereby in some embodiments, the CD200 antagonist is anti-CD200 antibody.As used herein, term " antibody " refers to can be in conjunction with complete antibody or the antibody fragment of CD200 or CD200R.Comprise Fab, Fv, scFv, Fab ' and F (ab ') ₂, mono-clonal and polyclonal antibody, through engineering approaches antibody (comprise antibody chimeric, strand, the CDR grafting, humanized, complete people, antibody with artificial selection) and the synthetic or the semisynthetic antibody that use phage display or alternative technology to produce.Also comprise with the several different methods through engineering approaches or produce with contain constant or Fc district variation or that change in conjunction with one or more effector cells' ability enhancing or weaken antibody; This type of variation antibody includes but not limited to such antibody, and wherein constant or Fc district contains the glycosylation pattern that changes.Small segment, as Fv and scFV because their small size and therefore outstanding tissue distribution and use for diagnosis and treatment and to have favorable properties.Have antibody constant or the Fc district through engineering approaches or variation and can be used to regulate effector function, as ADCC and CDC.This type of have through engineering approaches or the variation antibody constant or the Fc district can be used for such situation, wherein for example CD200 at normal tissue expression; In these situations, there is not the anti-CD200 antibody of the variation of effector function can cause that desirable treatment replys and do not destroy healthy tissues.In addition, antibody, variation antibody and its fragment can be closure (that is, described antibody or fragment suppress the interaction of CD200 and CD200R) or misclosure (that is, described antibody or fragment are attached to CD200 but do not block it and the interaction of CD200R).

The disclosure also relates to anti-CD200 antibody, and it comprises as heavy chain provided herein and light chain, comprises and heavy chain provided herein and/or light chain homology or similar heavy chain and light chain." homology " or " identity " or " similarity " refer between two peptides or two nucleic acid molecule between sequence similarity.Can determine homology and identity by the position of comparing in each sequence, can compare described sequence in order to compare purpose.Put when being occupied by identical base or amino acid when the sequence medium-priced that is compared, so described molecule is same in this position; When equivalent locations when occupying, is claimed that described molecule is homologous (similar) in this position by same or similar amino-acid residue (for example, similar in solid or charge property) so.The expression of per-cent homology/similarity or identity is meant same or similar amino acid no purpose function on the total position of the sequence that compared.The comparison based on mathematics of sequence similarity described in term " homology ", and it can be used to identify gene or the protein with identity function or motif.As used herein, " identity " refer to when the two or more sequences of comparison so that during sequences match maximization (promptly considering room and insertion), the per-cent of identical Nucleotide or amino-acid residue on the correspondence position in described sequence.Thereby the method for the definite identity of design (is seen Computational Molecular Biology, Lesk, A.M., ed., OxfordUniversity Press, New York, 1988 to obtain maximum match between the sequence of test; Biocomputing:Informatics andGenome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; With Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; And Carillo, H., and Lipman, D., SIAM J.Applied Math., 48:1073 1988, Devereux, J., wait the people, Nucleic Acids Research 12 (1): 387 (1984), BLASTP, BLASTN, FASTA (Altschul, S.F. wait the people, people Nuc.Acids Res.25:3389-3402 (1997) such as J.Molec.Biol.215:403-410 (1990) and Altschul) and BLAST X (BLAST Manual, Altschul, S., Deng the people, NCBI NLM NIH Bethesda, Md.20894; Altschul, S. waits the people, J.Mol.Biol.215:403-410 (1990).The sequence of " uncorrelated " or " non-homogeneous " is total less than 40% identity, although preferred and sequence of the present disclosure are less than 25% identity.In comparing two sequences, the existence of the disappearance of residue (amino acid or nucleic acid) or extra residue also reduces identity and homology/similarity.

Therefore, the disclosure relates to the antibody as herein described that does not have leader sequence.Thereby antibody of the present disclosure also comprises heavy chain and light chain (as described herein), and wherein leader sequence does not exist or replaced by different leader sequences.If produce antibody of the present disclosure, therefore can select suitable leader sequence according to used concrete host cell with host cell.

Can produce antibody by means commonly known in the art.For example, the fragment of using CD200 positive cell, CD200 polypeptide or CD200 polypeptide is as immunogen, thereby in animal, cause immunne response, can separate the cell that produces antibody and separate monoclonal antibody, can produce monoclonal anti CD200 antibody from this animal.The sequence of this antibody-like be can measure and antibody or its variant produced by recombinant technology.Can and can produce chimeric, the CDR grafting, humanized and complete people's antibody with recombinant technology based on monoclonal antibody in conjunction with the sequence of the polypeptide of CD200.

In addition, use the CD200 positive cell or can select antibody (" phage antibody ") from the reorganization library from its polypeptides derived, as bait with based on target specific isolation antibody or polypeptide.Inhuman and inosculating antibody CD200 production of antibodies and being separated in those skilled in the art's the limit of power.

Improve the antibody that produces in the inhuman cell with recombinant DNA technology.Thereby, can make up chimeric antibody so that reduce its immunogenicity in diagnosis or treatment application.In addition, modify humanized antibody by framework, can reduce immunogenicity by the CDR grafting is also optional.See U.S. Patent number 5,225,539, its content is incorporated herein by reference.

Antibody can obtain from animal serum, perhaps for monoclonal antibody or its segmental situation, produces in cell culture.Can be with recombinant DNA technology according to the method for setting up, the method that is included in bacterium or the preferred mammal cell cultures produces antibody.The preferred secretory antibody product of selected cell culture system.

In another embodiment, the method that is used to produce antibody disclosed herein comprises cultivates the host, for example, intestinal bacteria or mammalian cell, it transforms with the hybrid carrier.Carrier comprises one or more expression cassettes, and described expression cassette contains the promotor of first dna sequence dna that effectively is connected to the coded signal peptide, and described first dna sequence dna is connected to proteinic second dna sequence dna of encoding antibody with meeting frame.Collect then and separation antibody albumen.Randomly, expression cassette can comprise effectively be connected to the coding multiple antibody protein polycistron for example, the promotor of two cistron dna sequence dnas, described dna sequence dna meets frame ground separately and effectively is connected to signal peptide.

In suitable cell culture medium in external propagation of carrying out hybridoma or mammalian host cell, described substratum comprises conventional standard medium (as the Eagle substratum (DMEM) or the RPMI1640 substratum of Dulbecco improvement), optional additional mammalian blood serum (for example, foetal calf serum), perhaps trace element and growth are kept fill-in (as feeder cell, as normal mouse peritonaeum emigrated cell, splenocyte, bone marrow macrophage, 2-monoethanolamine, Regular Insulin, Transferrins,iron complexes, low-density lipoprotein, oleic acid, or the like).For the amplification of the host cell of bacterial cell or yeast cell is carried out in suitable culture medium known in the art equally.For example, for bacterium, suitable medium comprises substratum LE, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2 x YT or M9 minimum medium.For yeast, suitable medium comprises substratum YPD, YEPD, minimum medium or withdraws from substratum (Complete Minimal DropoutMedium) fully substantially.

Produced in vitro provides pure relatively antibody preparations and has allowed to amplify the antibody that production is wished in a large number.The technology that is used for bacterial cell, yeast, plant or mammalian cell cultivation is known in the art and (for example comprises the homogeneous suspension culture, in airlift reactor or continuous-stirring reactor), fixed or entrapped cell cultivate (for example, on tubular fibre, micro-capsule, agarose microballon or ceramic cartridge case).

By also obtaining a large amount of purpose antibody at the in-vitro multiplication mammalian cell.For this purpose, the hybridoma that produces purpose antibody is expelled in the Mammals of tissue compatible and causes the tumor growth that produces antibody.Randomly, before injection, use hydrocarbon, particularly mineral oil such as pristane (tetramethyl--pentadecane) cause animal.After 1 to 3 week, from those mammiferous body fluid separation antibodies.For example, the hybridoma that will obtain by suitable myeloma cell and cytogamy from the generation antibody of Balb/c mouse is perhaps taken office from the transfectional cell peritoneal injection of the generation purpose antibody of hybridoma cell line Sp2/0 and is selected for use in the pretreated Balb/c mouse of pristane.After 1 to 2 week, get ascites from animal.

The front with other technologies at for example Kohler and Milstein, (1975) Nature256:495-497; U.S. Patent number 4,376,110; Harlow and Lane, Antibodies:aLaboratory Manual discusses among (1988) Cold Spring Harbor, and the open of them all is incorporated herein by reference.Be used to prepare the superincumbent reference of technology of recombinant antibody molecule and at for example WO97/08320; U.S. Patent number 5,427,908; U.S. Patent number 5,508,717; Smith, 1985, Science, Vol.225, pp 1315-1317; Parmley and Smith, 1988, Gene 73, pp305-318; People such as De La Cruz, 1988, Journal of Biological Chemistry, 263 pp4318-4322; U.S. Patent number 5,403,484; U.S. Patent number 5223409; WO88/06630; WO92/15679; U.S. Patent number 5780279; U.S. Patent number 5571698; U.S. Patent number 6040136; People such as Davis, 1999, Cancer Metastasis Rev., 18 (4): 421-5; Taylor waits the people, Nucleic Acids Research 20 (1992): 6287-6295; People such as Tomizuka, Proc.Natl.Academy of Sciences USA 97 (2) (2000): describe among the 722-727.The content of all these reference is incorporated herein by reference.

The pair cell culture supernatant is screened purpose antibody, preferably passes through the immunofluorescence dyeing of CD200 positive cell, by immunoblotting, and by enzyme immunoassay, for example, sandwich assay method or spot assay method, or radioimmunoassay.

For the separation of antibody, can concentrate the immunoglobulin (Ig) in culture supernatant or the ascites, for example, by ammonium sulfate precipitation, water-absorbing material such as polyoxyethylene glycol are dialysed, filter by selective membrane, or the like.If necessary and/or wish, can be by conventional chromatography method, for example, gel-filtration, ion exchange chromatography, usefulness DEAE-Mierocrystalline cellulose chromatography and/or (immunity) affinity chromatography, for example, use from one or more polypeptide surfaces of CD200 positive cell line or with A or the proteic affinitive layer purification antibody of G.

The method that another embodiment provides the bacterial cell of preparation antibody of the anti-CD200 of secretion in suitable Mammals to be.For example, rabbit is used merging sample or CD200 polypeptide or its fragment immunity from positive tissue of CD200 or cell.The phage display library that structure produces from the rabbit of immunity and according to method well known in the art (as the multiple references that is incorporated herein by reference disclosed method) elutriation purpose antibody.

The hybridoma of secrete monoclonal antibody is also disclosed.Preferred hybridoma is that inheritance stability, secretion have desired specific monoclonal antibody described herein, and can activate by thawing and cloning from the culture of deep refrigeration again.

The method of the hybridoma cell line of the monoclonal antibody for preparing the anti-CD200 of secretion is provided in another embodiment.In the method, with suitable Mammals, for example, the Balb/c mouse is with one or more polypeptide or the antigen fragment of CD200 or use from one or more polypeptide or antigen fragment, the CD200 positive cell self of CD200 positive cell or contain the antigen vectors immunity of purified polypeptide as described.Will through the cell fast culture growth of the mammiferous generation antibody of immunity or with the cytogamy of suitable myeloma cell line.The clone is merged the hybrid cell that obtains, and selects the cell clone of secretion purpose antibody.For example, will be with splenocyte of the Balb/c mouse of CD200 positive chronic Lymphocytic leukemia (CLL) clone immunity and the cytogamy of myeloma cell line PAI or myeloma cell line Sp2/0-Ag 14.Then the hybrid cell of gained is screened secretion and clone's positive hybridoma cell of purpose antibody.

The method of preferred preparation hybridoma cell line is characterized in that at some months, passes through subcutaneous and/or peritoneal injection 10 in 2 to 4 months ⁶To 10 ⁷The CD200 positive cell line of individual cell several times, for example, 4 to 6 times, immune Balb/c mouse.Injection back 2 to 4 days is from the mice immunized extracting spleen cell and merging promotor at last, and there is down the cytogamy with myeloma cell line PAI in preferred polyoxyethylene glycol.Preferably, have an appointment 30% in the solution of the polyoxyethylene glycol of about 50% molecular weight about 4000 containing, the splenocyte of myeloma cell and three to two times of excessive immune mouses of hanging oneself merges.After the fusion, in suitable medium as previously described (replenish and select substratum, for example HAT substratum),, surpass the purpose hybridoma so that prevent normal myeloma cell's growth with the timed interval amplifying cells of rule.

Antibody and its fragment can be " chimeric ".Chimeric antibody and its Fab comprise the part from two or more different plant species (for example mouse and people).To have and wish that the variable region montage of specific mouse can produce chimeric antibody (for example, U.S. Patent number 4,816,567) in people's constant domain constant gene segment C.By this way, the non-human antibody can be modified so that they are more suitable for human clinical application.

Monoclonal antibody of the present disclosure comprises inhuman (for example mouse) antibody of " humanization " form.MAb humanized or the CDR grafting especially can be used as human therapeutical agent, because they equally with mouse antibodies are not removed from circulation and do not cause disadvantageous immune response usually.Usually, humanized antibody has the introducing one or more amino-acid residues from inhuman source wherein.These inhuman amino-acid residues are commonly referred to as " input " residue, and it is usually from " output " variable domains.The method for preparing humanized antibody is well known in the art.For example, basically according to Winter and colleague (people such as Jones, Nature 321:522-525 (1986); People such as Reichmann, Nature, 332:323-327 (1988); People such as Verheoeyen, Science, 239:1534-1536 (1988)) method, replace carrying out humanization by corresponding sequence with rodent CDR or CDR sequence personnel selection antibody.Also see people 2006 Mol Immunol 43:1243-1257 such as Staelens.In specific embodiments, the humanization form of inhuman (for example mouse) antibody is people's antibody (receptor antibody), and wherein the hypermutation of receptor antibody (CDR) district residue is replaced as the hypervariable region residue of the specificity with hope, avidity and the binding ability of mouse, rat, rabbit or non-human primates from inhuman species (donor antibody).In some cases, the framework region residue of human normal immunoglobulin is also replaced (being also referred to as " reverse mutation ") with corresponding inhuman residue.In addition, phage display library can be used to change the amino acid of selected location in the antibody sequence.Also can influence the form of humanized antibody by the selection of people's framework.In addition, can modify humanization and chimeric antibody to comprise the residue of in receptor antibody or donor antibody, not finding so that further improve antibody character, as avidity or effector function.

Fully human antibodies also is provided in the disclosure.Term " people's antibody " comprises that having from ethnic group is the antibody of the variable and constant region (if existence) of immunoglobulin sequences.People's antibody can comprise that can't help ethnic group is immunoglobulin sequences amino acids coding residue (for example, the sudden change that imports by somatic mutation at random external or site-specific mutagenesis or body).Yet term " people's antibody " does not comprise such antibody, wherein from another mammalian species, as the CDR sequence of the kind of mouse system by grafting (for example, humanized antibody) to people's frame sequence.Fully the people's or people's antibody can be from the transgenic mice (carry variable (V), diversity (D), connect (J) and constant (C) exon) of carrier's antibody gene or from people's cell.For example, may produce transgenic animal (for example mouse) now, its whole repertoires that can produce people's antibody when immunity when not producing endogenous immunoglobulin (Ig) (are for example seen people such as Jakobovits, PNAS, 90:2551 (1993); People such as Jakobovits, Nature, 362:255-258 (1993); People such as Bruggermann, Year inImmuno., 7:33 (1993); With people Nature 355:258 (1992) such as Duchosal.Can the through engineering approaches transgenic mice to contain gene order from the human immunoglobulin gene who does not reset.Human sequence can the encode heavy chain and the light chain of people's antibody, and will in mouse, correctly bring into play function, experience reset with provide to the mankind in similar wide antibody repertoire.Can use target protein (for example, CD200, its fragment are perhaps expressed the cell of CD200) immune transgenic mouse to produce different batches specific antibodies and their coding RNA.Then can with from the nucleic acid clone of the antibody chain component of this antibody-like of coding of animal in display carrier.Typically, the independent colony of the nucleic acid of clones coding heavy chain and sequence of light chain, separately colony recombinates when inserting in the carrier then, makes the carrier of any given copy accept the combination at random of heavy chain and light chain.Design vector makes them to be assembled on the outside surface of carrier-containing demonstration package and to show with the expressing antibodies chain.For example, antibody chain can be used as expressing fusion protein, and this fusion rotein has the bacteriophage coat protein from the phage outside surface.Afterwards, can be to the displaying of demonstration package screening in conjunction with the antibody of target.

In addition, people's antibody can be from phage display library (people such as Hoogenboom, J.Mol Biol, 227:381 (1991); People such as Marks, J.Mol.Biol., 222:581-597 (1991); People Nature Biotech 14:309 (1996) such as Vaughan).Can produce the synthetic phage library, it uses the combination at random in synthetic people antibody V district.By antigenic selection, can prepare fully human antibodies, wherein the V district is extraordinary image people's in nature.See patent US 6,794,132,6,680,209,4,634,666 and people (1983) such as Ostberg, Hybridoma 2:361-367 is incorporated herein by reference their content.

For people's production of antibodies, also see people Nature Genetics 15:146-156 (1997) such as Mendez, Green and Jakobovits J.Exp.Med.188:483-495 (1998) are incorporated herein by reference disclosing of they.People's antibody is further at United States Patent (USP) 5,939, discusses and describes in 598 and 6,673,986.Also see the United States Patent (USP) 6,114,598,6,075,181 and 6,162,963 that submit to June 5 nineteen ninety-five.Also see the United States Patent (USP) 6 that on October 2nd, 1996 submitted to, 150,584 and United States Patent (USP) 6,713,610 and 6,657,103 and U.S. Patent application 10/421,011 (US 2003-0229905A1), 10/455,013 (US 2004-0010810A1), 10/627,250 (US 2004-0093622A1), 10/656,623 (US 2006-0040363A1), 10/658,521 (US 2005-0054055A1), 10/917,703 (US 2005-0076395A1) and 10/978,297 (US 2005-0287630A1).Also see and authorize the international patent application no WO 96/34096 that the international patent application no WO that announces in european patent number EP 0 463 151 B1 that announce, on February 3rd, 1994 October 31 in 94/02602,1996 announced and the WO 98/24893 of announcement on June 11st, 1998 in the PCT/US93/06926 that submitted on July 23rd, 1993, on July 12nd, 1996.Open all complete being incorporated herein by reference with above-cited every piece of patent, application and reference.

In alternative approach, other people comprise GenPharm International, and Inc. has utilized " miniature locus " (minilocus) method.In miniature locus method, by fragment (genes of individuals) the simulation external source Ig locus that comprises the Ig locus.Thereby, one or more V _HGene, one or more D _HGene, one or more J _HGene, a μ constant region and another constant region (preferred γ constant region) are formed for being inserted into the construct in the animal.This method is at people's such as Surani U.S. Patent number 5,545,807 and the U.S. Patent number 5,545 of Lonberg and Kay, 806,5,625,825,5,625,126,5,633,425,5,661,016,5,770,429,5,789,650 and 5,814,318, the U.S. Patent number 5,591 of Krimpenfort and Bems, 669, people's such as Berns U.S. Patent number 5,612,205,5,721,367,5,789,215 and Choi and Dunn, with describe in the U.S. Patent number 5,643,763 of GenPharmInternational.Also see United States Patent (USP) 5,569,825,5,877,397,6,300,129,5,874,299,6,255,458 and 7,041,871,, disclosing of they is incorporated herein by reference.Also see european patent number 0 546 073 B1, international patent application no WO92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852 and WO98/24884 are incorporated herein by reference disclosing of they is complete.Also see people (1992Nuc.Acids.Res. such as Taylor, 20:6287), people such as Chen (1993 Int.Immunol.5:647), people such as Tuaillon (1993 PNAS U S A.90:3720-4), people such as Choi, (1993 NatureGenetics 4:117), people such as Lonberg (1994 Nature 368:856-859), people such as Taylor (1994 International Immunology 6:579-591), with people (1995 JImmunol.154:6453-65) such as Tuaillon, people such as Fishwild (1996 Nature Biotechnology 14:845), with people (2000 Eur J Immunol.10:2998-3005) such as Tuaillon, be incorporated herein by reference disclosing of they is complete.

In some embodiments, provide anti-CD200 antibody or its Fab that goes immunity.Can modify the antibody of immunity or its Fab so that make that this antibody or its Fab are non-immunogenic for given species, perhaps immunogenicity reduces.Utilize multiple technologies well known by persons skilled in the art can realize immunity (for example seeing PCT publication No. WO 04/108158 and WO 00/34317) by modified antibodies or its Fab.For example, interior potential t cell epitope and/or the B cell epitope of aminoacid sequence that is tested and appraised antibody or its Fab also for example uses recombinant technology to remove one or more potential t cell epitopes and/or B cell epitope from antibody or its Fab, antibody or its Fab can be gone immunity.Can choose wantonly then and produce and test modified antibody or its Fab and kept the biologic activity of one or more hope,, but have immunogenic antibody or its Fab that reduces as binding affinity with evaluation.The method of potential t cell epitope and/or B cell epitope of identifying can be carried out with technology known in the art, (for example see as computer approach, PCT publication No. WO 02/069232), technology on the external or computer chip, with bioassay method or physical method (as measuring combining of peptide and MHC molecule, measure peptide: MHC complex body and combining from the TXi Baoshouti of species to accept described antibody or its Fab, the transgenic animal that use is accepted described antibody or its Fab with the MHC molecule of described species are tested described protein or its peptide moiety, or test with transgenic animal, these transgenic animal are used from the immune system cell of described species and rebuild accepting described antibody or its Fab, or the like).In multiple embodiments, the anti-CD200 antibody of immunity that goes described herein comprises immune Fab, Fab, Fv, scFv, Fab ' and F (ab ') ₂, monoclonal antibody, murine antibody, through engineering approaches antibody (as the antibody of antibody, humanized antibody, fully human antibodies and the artificial selection of chimeric antibody, single-chain antibody, CDR grafting), synthetic antibody and semi-synthetic antibody.

In further embodiment, produced recombinant DNA, it comprises the insertion fragment of coding at the weight chain variable structural domain and/or the light chain variable structural domain of the antibody of CD200 or CD200 positive cell line.Term DNA comprises coding single stranded DNA, the double-stranded DNA of being made up of described coding DNA and its complementary DNA, or these complementary (strand) DNA self.

In addition, coding can be the DNA of enzymatic or chemosynthesis at the weight chain variable structural domain of the antibody of CD200 or CD200 positive cell line and/or the DNA of light chain variable structural domain, and it has true dna sequence dna or its mutant of encoding heavy chain variable domains and/or light chain variable structural domain.The mutant of true DNA is the weight chain variable structural domain of the above-mentioned antibody of coding and/or the DNA of light chain variable structural domain, and wherein one or more amino acid are lacked, insert or exchange with one or more other amino acid.Preferably, optimize in the application the described CDR outside that is modified at the weight chain variable structural domain and/or the light chain variable structural domain of antibody at humanization and expression.The term mutant DNA also comprises the silent mutation body, and wherein one or more Nucleotide are had other Nucleotide of Xinmi City's numeral of coding same amino acid and replace.The term mutant nucleotide sequence also comprises degenerate sequence.Degenerate sequence is a degeneracy in the genetic code meaning, does not cause the change of initial amino acid sequence coded because the Nucleotide of infinite number is replaced by other Nucleotide.Because their different restriction sites and/or specific host, the frequency of the preferred concrete codon of intestinal bacteria especially, this type of degenerate sequence can be used to obtain the optimum expression of heavy chain mouse variable domains and/or light chain mouse variable domains.

The term mutant is intended to comprise the dna mutation body that obtains according to the vitro mutagenesis of methods known in the art by true DNA.

For the assembling of complete tetramer immunoglobulin molecules and the expression of chimeric antibody, with the corresponding DNA fusion that the recombinant DNA of encoding heavy chain and light chain variable structural domain inserts fragment and encoding heavy chain and light chain constant domain, transfer in the proper host cell after for example being incorporated into hybrid vector then.

Can use and comprise the segmental recombinant DNA of insertion, described insertion fragment coding and people's constant domain IgG, for example γ 1, γ 2, γ 3 or γ 4; γ 1 or γ 4 merges in specific embodiments at the heavy chain mouse variable domains of the antibody of CD200 or CD200 positive cell line.Also provide to comprise and insert segmental recombinant DNA the light chain mouse variable domains at the antibody of clone disclosed herein of described insertion fragment coding and people's constant domain K or λ, preferred K fusion.

Another embodiment relates to the recombinant DNA of the recombinant polypeptide of encoding, wherein the weight chain variable structural domain is connected by spacer with the light chain variable structural domain, the optional peptide that signal sequence that enhancing antibody processes in host cell and/or coding make things convenient for antibody purification and/or the dna sequence dna of cleavage site and/or peptide transcribed spacer and/or promoting agent of comprising.The DNA of coding promoting agent means coding and can be used for diagnosing or treating the DNA of the promoting agent of application.Thereby, especially be designated as the active agent molecule of toxin or enzyme, activatory enzyme that particularly can the catalyged precursor medicine.The DNA of this promoting agent of encoding has the dna sequence dna of naturally occurring enzyme of coding or toxin, perhaps its mutant, and can prepare by means commonly known in the art.

Therefore, monoclonal antibody of the present disclosure or Fab can be naked antibody or the Fabs that is not conjugated to other materials such as therapeutical agent or certification mark.Alternatively, monoclonal antibody or Fab can be conjugated to material such as cytotoxic agent, small molecules, hormone, enzyme, somatomedin, cytokine, ribozyme, peptide mimics, chemical, prodrug, nucleic acid molecule, comprise that encoding sequence is (as antisense, RNAi, gene targeting construct, or the like), perhaps certification mark (as NMR or x-ray contrast agent, fluorescence molecule, or the like).In some embodiments, anti-CD200 polypeptide or Fab (for example, Fab, Fv, strand scFv, Fab ' and F (ab ') ₂) be connected to the molecule of the half life that increases described polypeptide or Fab.The molecule that can be connected to described anti-CD200 polypeptide or Fab includes but not limited to serum protein, comprises albumin, polypeptide, other protein or protein domain, and PEG.

Several possible carrier systems are used in the heavy chain and the light chain gene of cloning by expression in the mammalian cell.One class carrier depends on the integration of target gene sequences to the host cell gene group.Cell with DNA of stable integration can be by importing drug resistance gene such as intestinal bacteria gpt (Mulligan simultaneously, R.C. and Berg, P., Proc.Natl.Acad.Sci., USA, 78:2072 (1981)) or Tn5 neo (Southern, P.J. and Berg, P., J.Mol.Appl.Genet., 1:327 (1982)) select.Selectable marker gene can be connected to DNA gene order to be expressed, perhaps import identical cell (Wigler, people such as M., Cell, 16:77 (1979)) by cotransfection.The second class carrier utilization is given the DNA element of self-replicating ability to the outer plasmid of karyomit(e).These carriers can be from animal virus, as bovine papilloma virus (Sarver, people such as N., Proc.Natl.Acad.Sci., USA, 79:7147 (1982)), polyomavirus (Deans, people such as R.J., Proc.Natl.Acad.Sci., USA, 81:1292 (1984)), or SV40 virus (Lusky, M. and Botchan, M., Nature, 293:79 (1981)).

Because immunoglobulin (Ig) cDNA only comprises the sequence of representing the proteic ripe mRNA of encoding antibody, come synthetic immunoglobulin mRNA so need regulatory gene to transcribe the extra gene expression element of processing with RNA.These elements can comprise splicing signal, transcripting promoter, comprise inducible promoter, enhanser, and termination signal.The cDNA expression vector that mixes these elements comprises Okayama, H. and Berg, P., Mol.Cell Biol., 3:280 (1983); Cepko, people such as C.L., Cell, 37:1053 (1984); And Kaufman, R.J., Proc.NatL Acad.Sci., USA, 82:689 (1985) described those.

In some embodiments, anti-CD200 antibody can be closure or misclosure antibody.As used herein, blocking antibody can be an interactional antibody between blocking-up CD200 and the CD200R.The misclosure antibodies to and/or interact with CD200 but do not block the interaction of itself and CD200R.Thereby, in some embodiments, anti-CD200 antibody be closure or misclosure mouse, chimeric, humanized, the people's or remove the antibody of immunity.

II. the CD200 antagonist that has the effector function of change

The CD200 antagonist can be reformed to cause effect increase or that reduce with respect to initial or parent's antagonist.For example, can after being attached to CD200, cause subfunction, and in some cases, suppress CD200:CD200R and interact in conjunction with the antagonist of CD200.For example, antagonist can contain other parts extra binding site of (comprising acceptor or extracellular protein).Can cause other incidents with combining of these other parts, as the attraction of other cells or raise and the activation of (comprising necrocytosis) of multiple incident.Thereby in some respects, the disclosure relates to the CD200 antagonist of the subfunction (or being called effector function hereinafter) that causes change.In some embodiments, the CD200 antagonist with secondary or effector function of change demonstrates increase, that reduce or does not have secondary or effector function, and can block or not block CD200:CD200R and interact.In specific embodiment, the CD200 antagonist with secondary or effector function of change is anti-CD200 antibody.

A) effector function

Multiple the replying of interaction influence of antibody and antibody-antigenic complex and immune system cell is called effector function in this article.Exemplary effector function comprises downward modulation (for example, the B-cell receptor of Fc receptors bind, phagolysis, cell surface receptor; BCR), or the like.Other effector functions comprise ADCC, wherein the Fc acceptor of antibodies on natural killer (NK) cell or the scavenger cell causes necrocytosis, and CDC, it is the necrocytosis of activation inductive (Daeron, Annu.Rev.Immunol.15:203-234 (1997) by complement cascade; Ward and Ghetie, Therapeutic Immunol.2:77-94 (1995); With Ravetch and Kinet, summary among the Annu.Rev.Immunol.9:457-492 (1991)).This type of effector function usually needs and will and can use as multiple assay method disclosed herein and assess with the Fc district of binding domains (for example, antibody variable territory) combination.

Some antibody mediated effect subfunctions comprise ADCC, and by Fc acceptor (FcR) mediation, FcR is attached to the Fc district of antibody.In ADCC, NK cell or scavenger cell are attached to the Fc district of antibody complex body and promote the cracking of target cell.The crosslinked cytotoxicity that causes perforin/granzyme mediation of FcR on the NK cell, and in scavenger cell, this crosslinked promotion medium such as nitrogen protoxide (NO), TNF-α and other release of active oxygen.For the CD200 positive target cell, the anti-CD200 antibodies target cell of effector function being led to target cell and Fc district.Antibody is to the avidity of concrete FcR, thereby antibody-mediated effector activity can be regulated by the Fc of change antibody and/or the aminoacid sequence and/or the posttranslational modification of constant region.

FcR is by they specificity definition to the immunoglobulin (Ig) isotype; The Fc acceptor of IgG antibody is known as Fc γ R, for IgE, is known as Fc ε R, for IgA, is known as Fc α R or the like.Fc γ R:Fc γ RI (CD64), Fc γ RII (CD32) and the Fc γ RIII (CD16) of three subclass have been identified.Because each Fc γ R subclass is by two or three genes encoding, and selectivity RNA montage causes multiple transcript, so there is the highly diverse of Fc γ R isotype.Three kinds of genes of coding Fc γ RI subclass (Fc γ RIA, Fc γ RIB and Fc γ RIC) cluster in No. 1 chromosomal long-armed 1q21.1 district; Two kinds of genes of the gene of coding Fc γ RII isotype (Fc γ RIIA, Fc γ RIIB and Fc γ RIIC) and coding Fc γ RIII (Fc γ RIIIA and Fc γ RIIIB) all cluster in the 1q22 district.These different FcR hypotypes are expressed (summary among Ravetch and the Kinet, Annu.Rev.Immunol.9:457-492 (1991)) on different cell types.For example, in the mankind, only on neutrophilic granulocyte, find Fc γ RIIIB, and find Fc γ RIIIA at scavenger cell, monocyte, natural killer (NK) cell and T cell subsets.Notably, Fc γ RIIIA is that NK cell (participating in a kind of cell type of ADCC) is gone up the unique FcR that exists.

Fc γ RI, Fc γ RII and Fc γ RIII are immunoglobulin superfamily (IgSF) acceptors; Fc γ RI has three IgSF structural domains in its extracellular domain, and Fc γ RII and Fc γ RIII only have two IgSF structural domains in their extracellular domain.The Fc acceptor of another type is a newborn Fc acceptor (FcRn).FcRn is structurally similar with major histocompatibility complex (MHC) and be made up of the non-covalent α-chain that combines B2M.

The binding site to Fc γ R on people and the murine antibody was navigated to " the following hinge area " that is called in the past, this time hinge area is made up of (as people such as Kabat residue 233-239, Sequences of Proteinsof Immunological Interest, the 5th edition, Public Health Service, NationalInstitutes of Health, Bethesda, EU among the Md. (1991) numbering) people Molec.Immunol.23:319-330 (1986) such as .Woof; People Nature 332:563 (1988) such as Duncan; Canfield and Morrison, J.Exp.Med.173:1483-1491 (1991); People such as Chappel, Proc.Natl.Acad.Sci USA 88:9036-9040 (1991).For residue 233-239, P238 and S239 have been cited as and may have related to combination.

Other zones of quoting in the past of bonded that may relate to Fc γ R are: people Fc γ RI's is that G316-K338 (human IgG) (only compares by sequence; Assessment does not substitute mutant) people Molec Immunol.23:319-330 (1986) such as () Woof; The K274-R301 (human IgG1) (based on peptide) of people Fc γ RIII (people Molec.Immunol.21:43-51 (1984) such as Sarmay); The Y407-R416 (human IgG) (based on peptide) of people Fc γ RIII (people Biochem.Soc.Trans.12:739-743 (1984) such as Gergely); And the N297 of mouse Fc γ RII and E318 (mouse IgG2b) (people such as Lu nd, Molec.Immunol., 29:53-59 (1992)).

People effector cell is the white corpuscle of expressing one or more FcR and carrying out effector function.In some embodiments, cell expressing Fc γ RIII and carry out the ADCC effector function at least.The example of the human leukocyte of mediation ADCC comprises peripheral blood lymphocytes (PBMC), natural killer (NK) cell, monocyte, cytotoxic T cell and neutrophilic granulocyte.The effector cell can be from its natural origin, as separating from blood or PBMC.

In CDC, antibody-antigenic complex conjugated complement causes the activation of complement cascade and produces MAC.The activation of CCP is by first kind of component (C1q) of complement system and the initiation that combines of (the suitable subclass) antibody that is attached to its isoantigen; Thereby the activated partial of complement cascade is regulated by this immunoglobulin (Ig) and the proteic binding affinity of C1q.C1q and two kinds of serine protease C1r and C1s form complex body C1, and it is first kind of component of CDC approach.C1q is that molecular weight is about 460,000 sexavalence molecule, and six collagens " stem " are connected to six spherical head zones in its structure.Burton and Woof, Advances in Immunol-51:1-84 (1992).For the complement activation cascade, C1q must be attached to the IgG1, the IgG2 that are attached to the antigenicity target or at least two molecules of IgG3, but the IgM of a molecule (Ward and Ghetie, Therapeutic Immunology 2:77-94 (1995) are p.80) only.In order to assess complement activation, can carry out as people such as Gazzano-Santoro the CDC assay method of describing among the J.Immunol.Methods 202:163 (1996).

The a plurality of residues that proposed the IgG molecule relate to the combination to C1q, described residue comprises Glu318, Lys320 and Lys322 residue on the CH2 structural domain, be positioned at the approaching closely corner of identical β chain on amino-acid residue 331, be arranged in the Lys235 and the Gly237 residue of hinge area down, (for example see with the residue 231 to 238 of the N-end region that is arranged in the CH2 structural domain, people such as Xu, J.Immunol.150:152A (summary) (1993), WO94/29351; People such as Tao, J.Exp.Med., 178:661-667 (1993); People such as Brekke, Eur.J.Immunol, 24:2542-47 (1994); People such as Burton; Nature, 288:338-344 (1980); Duncan and Winter, Nature332:738-40 (1988); U.S. Patent number 5,648,260 and U.S. Patent number 5,624,821).Further propose, IgG also depends on existence, disappearance or modification (it is anchored on Asn297 usually) (Ward and Ghetie, the Therapeutic Immunology 2:77-94 (1995) of the carbohydrate part between two CH2 structural domains in conjunction with the ability of C1q and complement activation cascade.In some embodiments, these residues one or more can be modified, substituted or removed or can be inserted one or more amino-acid residues so that strengthen or reduce the CDC activity of anti-CD200 antibody provided herein.

B) has the anti-CD200 antibody of the effector function of being regulated

Can regulate the effector function of the constant region that relates to the target specific antibody by the character that changes constant or Fc district.The effector function that changes for example comprises one or more following active adjustings: ADCC, CDC, apoptosis, with one or more Fc acceptors combine and short inflammation is replied.Adjusting refers to compare with the activity of second kind of antibody, active increase, reduces, or eliminates.In some embodiments, second kind of antibody is the antibody with effector function.Antibody that second kind of antibody can be through engineering approaches or naturally occurring antibody and can refer to unmanifest, natural or parental antibody.In specific embodiments, regulate such situation that comprises, wherein activity is removed or lacks fully.In addition, in some cases, unmanifest antibody can demonstrate similar or be equal to the active effector function activity of chC2aB7-hG1 disclosed herein or hB7V3V2-hGl antibody.Equally, functional or unmanifest constant or Fc district can have the effector function of natural constant or Fc structural domain; In some cases, the constant or Fc district of chC2aB7-hGl or hB7V3V2-hGl can represent unmanifest structural domain.For this purpose, chC2aB7-hGl and hB7V3V2-hGl are other antibody activity comparative standards, and hB7V3V2-hG1 is preferred standard.

Have the FcR binding affinity of change and/or the ADCC active polypeptide variants of CDC active and/or that change and be and compare the FcR that has enhanced or reduce with natural or parent's polypeptide or the polypeptide that comprises native sequences Fc or constant region in conjunction with activity and/or ADCC activity and/or the active polypeptide of CDC.Demonstrate to the bonded polypeptide variants of the increase of FcR with than the bigger avidity of parent polypeptide in conjunction with at least a FcR.Demonstrate to the bonded polypeptide variants that reduces of FcR with than the lower avidity of parent polypeptide in conjunction with at least a FcR.This type of variant of the bonded that reduces that demonstrates FcR can have very little or imperceptible combination to FcR, and for example, or Fc district constant with the native sequences immunoglobulin (Ig) and FcR compare the combination of 0-20% in conjunction with level.Similarly, ADCC and/or the active polypeptide variants of CDC that demonstrates change compared the ADCC and/or the CDC activity that can demonstrate increase or reduce with natural or parent's polypeptide.The polypeptide variants that demonstrates the ADCC that reduces and/or CDC can demonstrate for example reduce or do not have ADCC as shown here and/or a CDC activity.In some embodiments, parent or natural polypeptides and its variant are antibody or Fab.In specific embodiments, described antibody or Fab are in conjunction with CD200 and can block or not block the CD200:CD200R interaction.

Native sequences Fc or constant region comprise with nature in the Fc that finds or the identical aminoacid sequence of aminoacid sequence of constant sequence.Fc variation or that change or constant region are because for example at least one is amino acid modified, insert or lack and comprise the different aminoacid sequence with native sequences heavy chain district.In some embodiments, variation or the constant region that changes are compared with the constant region of native sequences constant region or parent's polypeptide has at least one amino acid replacement, insertion and/or disappearance, for example, in the constant region of native sequences constant region or parent's polypeptide about one to about hundreds of amino acid replacement, insertion and/or disappearance.In some embodiments, the constant region of the variation of this paper or the constant region of change and native sequences constant region and/or parent's polypeptide has at least about 70% homology (similarity) or identity, in some cases, have with it at least about 75%, in other cases, at least about 80% homology or identity, in other embodiments, have with it at least about 85%, 90% or 95% homology or identity.Variation or the constant region that changes also can contain one or more aminoacid deletion or insertion.In addition, the constant region of variation can contain one or more amino acid replacements, disappearance or insertion, and it causes the posttranslational modification that changes, for example comprises the glycosylation pattern of change.

Can be as the anti-CD200 antibody of current disclosed variation by nucleic acid sequence encoding, this nucleic acid sequence encoding has one or more aminoacid insertion, disappearance or alternate polypeptide with respect to natural or parent's peptide sequence.In addition, variation antibody can be by nucleic acid sequence encoding, this nucleotide sequence under stringent condition with the nucleic acid array hybridizing of the anti-CD200 antibody of coding variation.Can use multiple condition to detect hybridization, mainly determine severity by the washing stage of hybridization assays method.Usually, high temperature and low salt concn obtain high severity, and low temperature and high salt concentration obtain low severity.Realize low strict hybridization by for example in about 2.0 x SSC, washing under 50 ℃, under 50 ℃, realize high severity with about 0.2 x SSC.

The antibody that has constant region, Fc or a heavy chain district of variation by through engineering approaches or generation can produce has antibody change or that do not have the effect subfunction or its Fab; Recombinant DNA technology and/or cell cultures and expression condition can be used to produce function and/or the active antibody with change.For example, recombinant DNA technology can be used for one or more amino acid replacements, disappearance or the insertion in the through engineering approaches zone (for example, Fc or constant region), and described regional effect antibody function comprises effector function.Alternatively, by operating so as to cell cultures that produces antibody and the change that expression condition can be realized posttranslational modification such as glycosylation pattern.

Therefore, aspects more of the present disclosure and method relate to the anti-CD200 antibody of the effector function with change, and it comprises one or more amino acid replacements, insertion and/or disappearance.In some embodiments, the anti-CD200 antibody of this variation demonstrate reduction or do not have an effect subfunction.In specific embodiments, variation antibody comprises the G2/G4 construct (for example seeing Figure 10,11,12,13 and 15) that replaces the G1 structural domain.

Except the exchange G1 structural domain and G2/G4 construct that provide as this paper, can produce the anti-CD200 antibody of effector function by the change of in some regional aminoacid sequence of antibody, introducing other types with reduction.This type of aminoacid sequence changes people such as including but not limited to Bluestone and (sees WO 94/28027 and WO 98/47531; Also see people 2000 Cell Immunol 200 such as Xu; 16-26) described Ala-Ala sudden change.Thereby in some embodiments, the anti-CD200 antibody that has sudden change (comprising the Ala-Ala sudden change) in constant region can be used for reducing or eliminating effector function.According to these embodiments, the constant region of anti-CD200 antibody comprises 234 sudden change and 235 sudden changes to L-Ala to L-Ala.In addition, constant region can contain two sudden changes: 234 sudden change and 235 second sudden changes to L-Ala to L-Ala.In one embodiment, anti-CD200 antibody comprises the IgG4 framework, wherein Ala-Ala sudden change will describe 234 from phenylalanine to the sudden change of L-Ala and/or 235 from the sudden change of leucine to L-Ala.In another embodiment, anti-CD200 antibody comprises the IgG1 framework, wherein Ala-Ala sudden change will describe 234 from leucine to the sudden change of L-Ala and/or 235 from the sudden change of leucine to L-Ala.Anti-CD200 antibody can be alternatively or is additionally carried other sudden changes, comprises point mutation K322A in CH2 structural domain people 2001 J Virol.75:12161-8 such as () Hezareh.The antibody that has described sudden change in constant region can still be closure or misclosure antibody.

Change in the hinge area also influences effector function.For example, the disappearance in the hinge area can reduce the avidity of Fc acceptor and can reduce complement activation (people 1981 PNAS USA78:524-528 such as Klein).Therefore the disclosure also relates to the antibody that has change in hinge area.

In specific embodiments, can modify anti-CD200 antibody to strengthen or to suppress to rely on the cytotoxicity (CDC) of complement.Can realize the CDC activity (for example seeing U.S. Patent number 6,194,551) of being regulated by in the Fc district of antibody, introducing one or more amino acid replacements, insertion or disappearance.Alternatively or extraly, can in the Fc district, import cysteine residues, thereby allow that the interchain disulfide linkage forms in this zone.The homodimer antibody of Chan Shenging can have raising or the internalization ability that weakens and/or the cell killing of complement-mediated increase or that reduce like this.See people such as Caron, J.ExpMed.176:1191-1195 (1992) and Shopes, B.J.Immunol.148:2918-2922 (1992), WO99/51642, Duncan ﹠amp; Winter Nature 322:738-40 (1988); U.S. Patent number 5,648,260; U.S. Patent number 5,624,821; And WO94/29351.Use assorted bi-functional cross-linking agent also can prepare homodimer antibody with enhanced anti-tumor activity as describing among the people Cancer Research 53:2560-2565 (1993) such as Wolff.Alternatively, can engineered antibody, thus it has two Fc district and has the cracking of enhanced complement and the ADCC ability.See people Anti-Cancer Drug Design 3:219-230 (1989) such as Stevenson.

Another possibility means of regulating the effector function of antibody comprise glycosylated change.By the Raju summary, it has summarized the importance of being advised (Raju, TS.BioProcess International April2003.44-53) of the oligosaccharides of finding to this theme on the human IgG of the effector function degree with them recently.According to Wright and Morrison, the microheterogeneity of human IgG oligosaccharides can influence biological function, as CDC and ADCC, with multiple Fc acceptor combine and with C1q combination of proteins (Wright A.﹠amp; Morrison SL.TIBTECH 1997,1526-32).The glycosylation pattern of write up antibody can depend on and produces cell with cell culture condition and different (Raju, TS.BioProcess International April 2003.44-53).This type of difference can cause change (the people Immunology.1996 such as Israel of effector function and pharmacokinetics; 89 (4): 573-578; People P.Clin.Exp.1996 such as Newkirk; 106 (2): 259-64).The difference of effector function can be attached to the ability of the Fc γ acceptor (Fc γ Rs) on the effector cell relevant with IgG.People such as Shields have shown, end user effector cell uses the bonded aminoacid sequence variant that Fc γ R is had raising, and IgG can demonstrate (the people JBiol Chem.2001 276 (9) such as Shields: 6591-604) up to 100% enhanced ADCC.Although these variants comprise the amino acid change of not finding at bonding interface, the character of sugar component and its structural pattern also can promote viewed difference.In addition, the existence of Fucose or disappearance can promote combination and ADCC (people J Biol Chem.2002 such as Shields in the oligosaccharide compositions of IgG; 277 (30): 26733-40).Lack and be connected to Asn ²⁹⁷The IgG of fructose carbohydrate demonstrate normal receptors bind to Fc γ acceptor.Compare, to Fc γ RIIA acceptor in conjunction with improving 50% and be accompanied by enhanced ADCC, especially under low antibody concentration.

People's such as Shinkawa work shows that the antibody of the anti-people IL-5 acceptor that produces demonstrates ADCC higher more than 50% (people J Biol Chem.2003 278 (5) such as Shinkawa: 3466-73) when comparing with the antibody of generation in Chinese hamster ovary cell (CHO) in big murine hybridoma.The IgG that monose is formed and the bright big murine hybridoma of oligosaccharides stave produces has the proteinic Fucose content that is lower than the CHO generation.The author infers that the shortage of the fucosylation of IgG1 has keying action in the active enhancing of ADCC.

People such as Umana adopt diverse ways, and it has changed glycosylation pattern (the people Nat Biotechnol.1999 Feb such as Umana of a kind of chimeric anti-neuroblastoma antibody of IgG1 chCE7; 17 (2): 176-80).Use tsiklomitsin, they have regulated the activity of glycosyltransferase (GnnII), and glycosyltransferase will be referred to the active oligosaccharides of ADCC to cutting.The ADCC activity of parental antibody only is higher than background level.The active measurement of ADCC of the chCE7 that produces under different tsiklomitsin levels has shown the optimum range that is used to obtain the active GnTIH expression of the maximum external ADCC of chCE7.The level to the complex oligosaccharide cut that this activity is relevant with constant region is relevant.Recently the variant of You Huaing demonstrates substantive ADCC activity.Similarly, Wright and Morrison have produced antibody (1994 J Exp Med 180:1087-1096) and shown that the antibody that produces can not carry out the lysis of complement-mediated in this clones in the Chinese hamster ovary celI system of glycosylation defect.Thereby since known to the change that influences effector function comprise the modification of glycosylation pattern or the change of glycosylated residues number, the disclosure relates to CD200 antibody, wherein glycosylation is changed to strengthen or to weaken effector function, comprises ADCC and CDC.The glycosylation that changes comprises the minimizing of glycosylated residues number or the change of increase and glycosylated residues pattern or position.

There is the additive method that changes the antibody mediated effect subfunction.For example, the cell that produces antibody can be high mutagenicity, has the Nucleotide of randomly changing and the antibody of polypeptid residue (seeing WO 2005/011735) in the whole antibody molecule thereby be created in.High mutagenicity host cell comprises dna mismatch rectification of defects cell.The antigenicity of the antibody of Chan Shenging can be lower and/or has useful pharmacokinetics character by this way.In addition, can select the character of this antibody-like, as enhanced or the effector function that weakens.

Also understanding effector function can become according to the binding affinity of antibody.For example, compare with the antibody with relatively low avidity in the complement activation system can more effective (people 1999 Immunol Invest 28:89-101 such as Marzocchi-Machado) for the antibody with high-affinity.Therefore, can change antibody make to its antigenic binding affinity reduce (for example, by such as one or more amino-acid residues substitute, add or the method for disappearance changes the variable region of antibody).Anti-CD200 antibody with binding affinity of reduction can demonstrate the effector function that weakens, and for example comprises ADCC that weakens and/or CDC.

III. exhaust or removed the method for the cell of expressing CD200

According to the disclosure, provide by the experimenter being used the therapy that comprises the CD200 antagonist to exhaust the method for expressing the cell of CD200 among the experimenter.As mentioned above, CD200 expresses on some immunocyte; And as illustrating in the disclosure, CD200 also expresses on some malignant cell.This differential expression of CD200 provides the therapy target to decide the approach of cancer cells (as the CD200 positive cell).Equally, in the method for treatment autoimmune disease, can target decide CD200 positive immunocyte and be used to exhaust.

CD200 by with medullary cell on CD200R interact, by sending the inhibition Signal Regulation immunosuppression that is used for the active and/or migration of marrow sample.For example, the CD200 knock-out mice shows more activated immunne response people Science 2000 such as () Hoek after immunogenicity stimulates, and the cell of expressing CD200 is by inducing skew to cause immunosuppression (seeing data shown in this article) in the cytokine spectrum of the immunocyte of irriate.Especially, the CD200 positive cell can the inducing cell factor be produced from the skew of Th1 to Th2 in blended cell colony assay method.Although the CD200 positive cell can suppress immunne response, therefore, the CD200 positive cancer cell can be escaped immunocyte and attack.Yet the expression of CD200 on the film of cancer cells and immunocyte can be used at therapy fixed these cells that hit.For example, anti-CD200 antagonist can be decided the CD200 positive cell and destroy CD200:CD200R to interact by specific target, thereby suppresses immunosuppression, and with the positive cell targeted immune effector cell of CD200.Therefore, embodiment of the present disclosure relates to target and decides the method that the CD200 positive cell is used to exhaust, and this method comprises the antagonist in conjunction with CD200, and in some embodiments, destroys CD200:CD200R and interact.

In some embodiments, the disclosure relates to the method that enhancing immunity is replied.These class methods comprise uses the therapy that comprises the CD200 antagonist, and in specific embodiments, antagonist is anti-CD200 antibody or the Fab that provides as this paper.Do not wish by any concrete mechanism constraint, remove the CD200 positive cell thereby the anti-CD200 antibody of closure, Fab, polypeptide or other antagonists can suppress to allow immunocyte to attack and remove the CD200 positive cell by blocking immunity.Alternatively or with aforementioned mechanism combine, anti-CD200 antibody (closure or misclosure) or other antagonists can be raised effector cell or other parts (for example complement component) described antibody or antagonist bonded CD200 positive cell and target and decide the necrocytosis of CD200 positive cell with the mediation of generating effect.

On the one hand, the disclosure relates to mouse, chimeric by its experimenter of needs is used, humanized or human anti cd 20 0 antibody is regulated the ADCC of CD200 positive target cell and/or the method for CDC.The disclosure relates to the ADCC that causes increase and/or the anti-CD200 antibody of variation of CDC, also relates to demonstrating the anti-CD200 antibody that reduces or do not have ADCC and/or the active variation of CDC.

In one embodiment, the anti-CD200 antibody of variation comprises Fc or constant region variation or that change, and wherein Bian Yi Fc or constant region demonstrate the enhanced effector function.This type of described region of variability can contain one or more amino acid replacements, insertion or disappearance.Alternatively or extraly, the posttranslational modification that Fc variation or that change or constant region can comprise change for example comprises the glycosylation pattern of change.The glycosylation pattern that changes comprises the modification of the increase of glycosidic link number or the position of minimizing and/or one or more glycosidic links (as the amino-acid residue number).

In another embodiment, the disclosure relates to the method that exhausts or remove the CD200 positive cell, and this method comprises and demonstrates the anti-CD200 antibody that reduces or do not have ADCC and/or the active variation of CDC.In one embodiment, the anti-CD200 antibody of variation comprises Fc or constant region variation or that change, wherein Bian Yi Fc or constant region demonstrate weaken or do not have an effect subfunction.Fc or constant region described this type of variation or that change can contain one or more amino acid replacements, insertion or disappearance.Alternatively or extraly, the Fc of variation or constant region can comprise the posttranslational modification of change, include but not limited to the glycosylation pattern of change.The example of the glycosylation pattern that changes is above being described.

In another embodiment, be applied to patient's mouse, chimeric, humanized or people or go the immunity anti-CD200 antibody be non-blocking antibody.The anti-CD200 antibody of misclosure can be as above-mentioned variation antibody and can therefore demonstrate the effector function of being regulated.For example, the anti-CD200 antibody of variation can not blocked the CD200:CD200R interaction and also can comprise the variation constant region that causes enhanced effector function such as enhanced ADCC.

A) treatment suffers from the patient's of autoimmune disease method

In some respects, the disclosure relates to the patient who suffers from autoimmune disease with the therapy for treating that comprises the CD200 antagonist.In some embodiments, described antagonist is anti-CD200 antibody or its Fab.In other embodiments, anti-CD200 antibody or its fragment are the anti-CD200 antibody that demonstrates the variation of the effector activity of being regulated.For example, variation antibody can comprise and can cause and increase or the variation of enhanced effector function such as ADCC or the constant region of change.In addition, described antibody can be non-blocking antibody and can be mouse, chimeric, humanized or people or remove the anti-CD200 antibody of immunity.Thereby patient's the method that treatment suffers from autoimmune disease can comprise any CD200 antagonist and the antibody as providing in the disclosure.

In some embodiments, anti-CD200 antibody or CD200 antagonist can be used to exhaust the cell in arbitrary type of its surface expression CD200, comprise for example immunocyte, as T cell, B cell and dendritic cell.In one embodiment, anti-CD200 antibody can be used for the directed immunocyte that destroys, and described immunocyte relates to undesired immunne response, as the immunne response relevant with autoimmune disease, transplanting, transformation reactions or inflammatory conditions.Can for example comprise that inflammatory response as inflammatory skin disease, comprises psoriatic and dermatitis (for example, atopic dermatitis) with the exemplary autoimmune disease and the illness of anti-CD200 Antybody therapy provided herein; Dermatomyositis; Systemic sclerosis and sclerosis; Relevant with inflammatory bowel reply (as crohn and ulcerative colitis); Respiratory distress syndrome (comprises adult respiratory distress syndrome; ARDS); Dermatitis; Meningitis; Encephalitis; Uveitis; Colitis; Glomerulonephritis; The allergy situation is as eczema and asthma and other situations and other inflammatory responses of relating to the infiltration of T cell; Atherosclerosis; Leukocyte adhesion deficiency; Rheumatoid arthritis; Systemic lupus erythematous (SLE); Diabetes (for example, the diabetes of type i diabetes or dependence Regular Insulin); Multiple sclerosis; Reynaud ' s syndrome; Autoimmune thyroiditis; Allergic encephalitis; Sjogren syndrome; Juvenile diabetes; With the cytokine of usually in pulmonary tuberculosis, sarcoidosis, polymyositis, granulomatosis and vasculitis, finding and the T cell mediated and immunne response acute and that delayed type hypersensitivity is relevant; Pernicious anemia (Addison disease); Relate to the disease that white corpuscle oozes out; Central nervous system (CNS) inflammatory conditions; Multiple organ injury's syndrome; Hemolytic anemia (including but not limited to the positive anaemia of cryoglobinemia or Coombs); Myasthenia gravis; The disease of immune complex mediation; Anti-glomerular basement membrane disease; Antiphospholipid syndrome; Allergic neuritis; Graves disease; Lan-Yi myasthenic syndrome; Bullous pemphigoid; Pemphigus; Autoimmune polyendocrine disease; The Josef Reiter disease; The stiff man syndrome; Behcet disease; Giant cell arteritis; Immune complex nephritis; IgA nephropathy; The IgM polyneuropathy; Immunologic thrombocytopenic purpura (ITP) or AT and autoimmune hemolytic, Hashimoto thyroiditis or the like.

According to method and composition as herein described, the disclosure also relates to the method for the treatment of transplanting or allotransplantation patient.Can transplant or the allogeneic program before or after the program patient used anti-CD200 antibody of the present disclosure or other CD200 antagonists so that reduce or remove the CD200 positive cell, described cell can reduce the acceptance of patient to transplanted organ or tissue.In specific embodiments, the transplant patient had the anti-CD200 antibody of enhanced effector function.In addition, anti-CD200 antibody is non-blocking antibody.

Can in combination treatment, use the therapy that comprises CD200 antagonist or antibody to the patient.Therefore, a part that the can be used as combination treatment orientation of using the immunocyte of some colony is killed and wounded and is used for the treatment of or prevents autoimmune disease, enhancing or postpone graft survival, treatment or prevention transformation reactions, perhaps treatment or prevention of inflammatory conditions.For example, the patient who accepts to comprise first kind of therapy of CD200 antagonist (for example, anti-CD200 antibody as herein described) also can be given second kind of therapy.The CD200 antagonist can be given simultaneously with second kind of therapy.Alternatively, the CD200 antagonist can be given before or after second kind of therapy.Second kind of therapy includes but not limited to anti-inflammatory agent, immunosuppressor and/or anti-infection agent.

Combination treatment of the present disclosure for example comprises, CD200 antagonist as described herein, and itself and steroid, antimalarial drug, acetylsalicylic acid, nonsteroid anti-inflammatory drugs, immunosuppressor or cytotoxic drug are used simultaneously or sequentially.Comprise that reflunomide (for example, prednisone, dexamethasone and prednisolone), methotrexate, methylprednisolone, macrolide immunosuppressant (for example, sirolimus and tacrolimus), mitotic inhibitor (for example, azathioprine, endoxan and methotrexate), the fungal metabolite that suppresses the T lymphocyte activity (for example, S-Neoral), mycophenlate mofetil, acetate glatiramer you and cytotoxic agent and DNA disrupting agent (for example, Chlorambucil).For autoimmune disorder and allogeneic or transplant patient, anti-CD200 therapy can make up with Antybody therapy, described Antybody therapy comprises daclizumab---a kind of human IgG1's monoclonal antibody of genetic modification, the α chain of its specific combination interleukin-2 acceptor, and multiple other targets are decided the antibody of immunocyte or other cells.This type of combination treatment can be used for the treatment of type 1 diabetes, rheumatoid arthritis, lupus and idiopathic thrombocytopenic purpura and other autoimmunization indications.The disclosure also relates to autoimmune disorder and transplant patient's therapy, and it comprises the CD200 antagonist (for example, the antibody described in the disclosure and its fragment) that is conjugated to one or more materials.

B) treatment cancer patients's method

On the one hand, the disclosure provides the treatment method for cancer, wherein the patient is used the promoting agent that destroys or suppress CD200 and its acceptor interaction.The interactional destruction of CD200:CD200R is reversed subsequently or is suppressed immunosuppression, thereby enhancing immunity is replied.Be used to destroy the interactional possible promoting agent of CD200:CD200R and for example comprise small molecules, chemical, polypeptide, inorganic molecule, and organometallic compound.Also can suppress CD200:CD200R by antisense, RNAi or gene therapy minimizing membranin or its receptor expression interacts.In addition, to CD200 or the special polypeptide of CD200R, can suppress the interactional immunosuppressive effect of CD200:CD200R as anti-CD200 or anti-CD200R specific antibody or its fragment.

Comprise any cancer cells that demonstrates CD200 expression or CD200 rise by the treatable cancer cells of CD200 antagonist.The cancer that anti-CD200 therapy can be used for the treatment of for example comprises, ovarian cancer, melanoma, myelomatosis, neurocytoma, kidney, mammary cancer, prostate cancer, hematologic malignancies (for example, lymphoma and leukemia) and plasmocyte cancer.Also comprise any cancer cells from neural crest cell.In some embodiments, the CD200 antagonist is anti-CD200 antibody.Can disturb the interaction of CD200 and its acceptor as this type of antibody of anticancer therapeutic agent.This interaction can be blocked the immunosuppressive effect of CD200.By improving immunne response by this way, this antibody-like can promote the removing of cancer cells.Anti-CD200 antibody also can be decided the necrocytosis that cancer cells is used for the effector mediation by target.

In one embodiment, can be applied to experimenter with demonstrating ADCC and/or the anti-CD200 antibody of the active variation of CDC regulated with CD200 positive cancer cell.For example, the anti-CD200 antibody of variation that is used for cancer therapy is compared with parent or natural antibody and can be demonstrated the enhanced effector activity.In another embodiment, the anti-CD200 antibody that makes a variation demonstrates the effector function that weakens with respect to natural antibody, comprises the ADCC that weakens.Described antibody can be mouse, chimeric, humanized, the people's or he goes the immunity antibody.The cancer that the anti-CD200 antibody that makes a variation can be used for the treatment of includes but not limited to the neural crest cell cancer.Also comprise plasmocyte cancer, ovarian cancer, skin carcinoma, lung cancer, kidney, mammary cancer, prostate cancer, neuroblastoma, lymphoma, myelomatosis and leukemia.

This antibody can be applied to the cancer patients as therapeutical agent, particularly but be not limited to, have CLL, plasmocyte cancer, ovarian cancer, skin carcinoma, lung cancer, kidney, mammary cancer, prostate cancer, neuroblastoma, lymphoma, myelomatosis, leukemia and from the patient of any cancer of neural crest cell.In the embodiment that is particularly useful, comprise that according to cancer therapy of the present disclosure (1) use anti-CD20 antibodies or antagonist, it disturbs the interaction between CD200 and its acceptor to suppress with blocking immunity, thereby promotes the removing of cancer cells; And/or (2) use fusion molecule, and it comprises that CD200 target certain portions is with direct kill cancer cell.Alternatively, antibody is by the direct kill cancer cell of cytotoxicity complement-mediated or that rely on antibody.Because CD200 also expresses on normal cell such as endotheliocyte, although be lower than the expression level on cancer cells, but also advantageously use anti-CD200 antibody, it has modified to reduce or to eliminate ADCC or CDC with the constant region of restriction to Normocellular injury.For example, if CD200 (for example is expressed in some activatory normal cells, the activated T cell) upward raised, make the kill and wound sensitivity of this type of cell to anti-CD200 antibody with effector function, therefore, also can advantageously use lack effector function anti-CD200 antibody to avoid helping exhausting of tumoricidal these cells.

In specific embodiments, by the IgG1 constant domain being exchanged for the effector function that anti-CD200 antibody is eliminated in IgG2/4 fusion structure territory.It is contemplated that the additive method of eliminating effector function, for example, it is known with the interactional site of FcR or insert peptide in hinge area suddenly change, thereby eliminates the required critical sites of FcR interaction.Anti-CD200 antibody with variation reduction or that do not have the effect subfunction also comprises the variant as former description.

Be used for inhibition or stop the interactional aforementioned substances of CD200:CD200R to use with other therapies or with other combinations of substances.Other materials include but not limited to polypeptide, small molecules, chemical, metal, organometallic compound, mineral compound, nucleic acid molecule, oligonucleotide, aptamers, spiegelmers, antisense nucleic acid, locked nucleic acid (LNA) inhibitor, peptide nucleic acid(PNA) (PNA) inhibitor, immunomodulator, Fab, prodrug and peptide simulated compound.

In some respects, the disclosure relates to combined therapy, and it comprises the CD200 antagonist, comprises compound as herein described and immunomodulatory compounds, vaccine or chemotherapy.The property the illustrated example that can be used for the suitable immunomodulator of this type of combination treatment comprise blocking t cell or antigen presenting cell negative adjusting material (for example, anti-CTLA 4 antibody, anti-PD-L1 antibody, anti-PDL-2 antibody, anti-PD-1 antibody or the like) or strengthen the T cell positive common stimulation material (for example, anti-cd40 antibody or anti-4-1BB antibody) or increase the material (for example, only anti-CD200 antibody or with the combination of inhibitor such as ImiDs, thalidomide or thalidomide analogue) of NK cell count or T cytoactive.In addition, the immunomodulatory therapy can comprise cancer vaccine, as load dendritic cell, protein, peptide, RNA or the DNA of tumour cell from this type of cell, the heat shock protein(HSP) in patient source (hsp ' s) or the general adjuvant of stimulating immune system on different levels, as any other compound of the acceptor of CpG, Luivac, Biostim, Ribominyl, Imudon, Bronchovaxom or activation innate immune system or other adjuvants (for example, toll sample receptor stimulant, anti-CTLA-4 antibody, or the like).And the immunomodulatory therapy can comprise with cytokine such as IL-2, GM-CSF and IFN-γ treatment.

In extra embodiment, before beginning anti-CD200 treatment, remove existing regulatory T cells with reagent such as anti-CD25, fludarabine or endoxan.And, strengthen marrow by anti-CD200 therapy and eradicate therapy, then be bone marrow transplantation or the therapeutic efficiency that shifts with the adaptability of the T cell of CLL cell response.In other embodiments, by suppressing the effect that mechanism improves anti-CD200 treatment with material such as anti-PDL1 and/or 2 antibody, anti-IL-10 antibody, anti-IL-6 antibodies or the like blocking immunity.In addition, can advantageously remove the Plasmacytoid dendritic cell, show that the Plasmacytoid dendritic cell are immunosuppressant in the cancer environment.The expection of sending at anti-CD200 antibody suppresses by blocking immunity for example, also can use the anti-CD200 antibody of the variation that lacks effector function in these embodiments that enhancing immunity replys.

In the embodiment that is particularly useful, the therapy that enhancing immunity is replied is separately or combines the polypeptide of CD200 with one of the immunomodulatory therapy of mentioning in the past combined administration.Therefore, CD200 antagonist (comprising anti-CD200 antibody as described herein) can with monoclonal antibody (for example, sharp appropriate uncommon agate, Si Tumanbu, A Lun pearl monoclonal antibody, Cetuximab or rhuMAb-VEGF), comprise that the monoclonal antibody (for example, gemtuzumab, ozogamicin, ibritumomab tiuxetan or tositumomab) of puting together is used in combination.

In addition, anti-CD200 therapy and chemotherapeutic combination especially can be used for alleviating total tumor load, the tumour accessibility takes place, strengthens, strengthen the susceptibility to ADCC with the restriction blood vessel, by providing more tumour antigen to cause the enhanced immunologic function, perhaps increase the expression of T cell attractive substance LIGHT.In the time will resisting the combination of CD200 therapy and another conventional antineoplastic agent to be applied to the experimenter simultaneously or sequentially, can show that anti-CD200 therapy strengthens the only result of treatment of arbitrary material.The medical compounds that can be used for the associativity antitumor therapy comprises (only illustrating): aminoglutethimide, amsacrine, Anastrozole, asparaginase, bcg, bicalutamide, bleomycin, buserelin, busulfan, camptothecine, capecitabine, carboplatin, carmustine, Chlorambucil, cis-platinum, CldAdo, clodronate disodium, colchicine, endoxan, cyproterone, cytosine arabinoside, Dacarbazine, gengshengmeisu, daunorubicin, Dienestrol, stilboestrol, docetaxel, Zorubicin, pidorubicin, estradiol, estramustine, Etoposide, Exemestane, filgrastim, fludarabine, fluohydrocortisone, Fluracil, Fluoxymesterone, Flutan, gemcitabine, genistein, Coserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, Interferon, rabbit, irinotecan, letrozole, folinic acid, leuprorelin acetate, LEVAMISOLE HCL, chlorethyl cyclohexyl nitrosourea, mustargen, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, Ortho-para-prism DDD, mitoxantrone, Nilutamide, the Luo Keda azoles, Sostatin, oxaliplatin, taxol, Pamidronate, pentostatin, Plicamycin, porfimer sodium, procarbazine, Raltitrexed, sharp appropriate uncommon agate, U-9889, Suramine, tamoxifen, Temozolomide, teniposide, testosterone, Tioguanine, thio-tepa, titanocene dichloride, topotecan, Si Tumanbu, tretinoin, vinealeucoblastine(VLB), vincristine(VCR), vindesine and vinorelbine.

These chemotherapy antineoplastic compound can be according to their mechanism of action grouping, for example comprise, the material of following classification: metabolic antagonist/carcinostatic agent, as pyrimidine analogue (5 FU 5 fluorouracil, floxuridine, capecitabine, gemcitabine and cytosine arabinoside) and purine analogue, antifol and relevant inhibitor (mercaptopurine, Tioguanine, pentostatin and 2-chlorodeoxyadenosine (CldAdo)); Antiproliferative/antimitotic agent, comprise natural product, as vinca alkaloids (vinealeucoblastine(VLB), vincristine(VCR) and vinorelbine), the microtubule disrupting agent is as Taxan (taxol, docetaxel), vincristine(VCR), vinealeucoblastine(VLB), the Luo Keda azoles, esperamicin and nvelbine, epidipodophyllotoxins (Etoposide, teniposide), DNA disrupting agent (actinomycin, amsacrine, the anthracene nucleus class, bleomycin, busulfan, camptothecine, carboplatin, Chlorambucil, cis-platinum, endoxan, endoxan, gengshengmeisu, daunorubicin, Zorubicin, pidorubicin, hexamethyl melamine, oxaliplatin, ifosfamide, melphalan, mustargen, mitomycin, mitoxantrone, nitrosourea, Plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethio-hosphopramide and Etoposide (VP16)); Microbiotic is as gengshengmeisu (dactinomycin), daunorubicin, Zorubicin (adriamycin), idarubicin, anthracene nucleus class, mitoxantrone, bleomycin, Plicamycin (Plicamycin) and mitomycin; Enzyme (altheine enzyme, its systemic metabolism altheine also exhausts the cell of the l-asparagine that can not synthesize self); Anti-platelet agents; Antiproliferative/antimitotic alkylating agent is as mustargen (mustargen, endoxan and analogue, melphalan, Chlorambucil), ethyleneimine and methyl melamine (hexamethyl melamine and thio-tepa), alkylsulfonate-busulfan, nitrosoureas (carmustine (BCNU) and analogue, U-9889), trazenes-Dacarbazine (DTIC); Antiproliferative/antimitotic metabolic antagonist is as folacin (methotrexate); Platinum coordination complex (cis-platinum, carboplatin), procarbazine, hydroxyurea, Ortho-para-prism DDD, aminoglutethimide; Hormone, hormone analogs (oestrogenic hormon, tamoxifen, Coserelin, bicalutamide, Nilutamide) and aromatase inhibitor (letrozole, Anastrozole); Anticoagulant (other inhibitor of heparin, synthetic heparinate and zymoplasm); Fibrinolytic agent (as tissue plasminogen activator, streptokinase and urokinase), acetylsalicylic acid, Dipyridamole, ticlopidine, clopidogrel, ReoPro; Anti-migration agent; Secretion inhibitor agent (breveldin); Immunosuppressor (S-Neoral, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenlate mofetil); (thalidomide and its analogue are as Revlimid (lenalidomide) (Revlimid, CC-5013) and CC-4047 (Actimid)), endoxan for immunomodulator; Angiogenesis inhibitor compound (TNP-470, genistein) and growth factor receptor inhibitors (vascular endothelial growth factor (VEGF) inhibitor, fibroblast growth factor (FGF) inhibitor); Angiotensin receptor blocker; Nitric oxide donors; Antisense oligonucleotide; Antibody (Si Tumanbu); Cell cycle inhibitor and differentiation inductor (tretinoin); MTOR inhibitor, topoisomerase enzyme inhibitor (Zorubicin (adriamycin), amsacrine, camptothecine, daunorubicin, gengshengmeisu, eniposide, pidorubicin, Etoposide, idarubicin and mitoxantrone, topotecan, irinotecan), reflunomide (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone and prednisolone); Growth factor signal transduction kinase inhibitor; Mitochondria dysfunction inductor and Caspase activator; With the chromatin disrupting agent.

In some embodiments, the medical compounds that can be used to make up the angiogenesis inhibitor therapy comprises: (1) " blood vessel generation molecule " is as bFGF (Prostatropin) release inhibitor; (2) neutralizing agent of blood vessel generation molecule is as anti-β bFGF antibody; (3) endotheliocyte of blood vessel generation stimulation is replied inhibitor, comprise collagenase inhibitors, basement membrane more novel inhibitors, angiostatic steroid, the angiogenesis inhibitor of originated from fungus, platelet factor 4, thrombospondin, arthritis drug, as Beracilline and mercaptosuccinic acid gold, vitamins D ₃Analogue, interferon-alpha, or the like.To the angiogenesis inhibitor of extra proposition, see people such as Blood, Biochim.Biophys.Acta, 1032:89-118 (1990), people such as Moses, Science, 248:1408-1410 (1990), people such as Ingber, Lab.Invest, 59:44-51 (1988) and U.S. Patent number 5,092,885,5,112,946,5,192,744,5,202,352 and 6,573,256.In addition, having multiple compound can be used to suppress blood vessel takes place, as Methionin binding fragment, the melanochrome of the peptide of the blood vessel generation approach of blocking VEGF-mediation or material, endostatin albumen or derivative, angiostatin or promote melanic compound, proplasmin fragment (for example, the Kringles1-3 of proplasmin), troponin subunit, vitronectin α _vβ ₃Antagonist, peptide, microbiotic or analogue from Saposin B (for example, tsiklomitsin, perhaps Xin Meisu), contain dienogest composition, be coupled to compound, compd E M-138, cinnamophenone and its analogue and the naaladase inhibitor that MetAP-2 suppresses core that comprise of peptide.For example see U.S. Patent number 6,395,718,6,462,075,6,465,431,6,475,784,6,482,802,6,482,810,6,500,431,6,500,924,6,518,298,6,521,439,6,525,019,6,538,103,6,544,758,6,544,947,6,548,477,6,559,126 and 6,569,845.

The character that depends on the composition therapy can continue using of anti-CD200 antibody and uses and/or use afterwards other therapies simultaneously.Using of antibody can be carried out with single dose or multidose.In some cases, before routine treatment, began using of anti-CD200 antibody at least in several days, and in other cases, before routine treatment is used at once or began at that time to use.In some cases, anti-CD200 antibody will be used after other therapies, and perhaps it can alternately be used with other therapies.

Antibody of the present invention can be used for directly killing or removing in vivo cancerous cells.Directly kill the experimenter's administration of antibodies (its optional cytotoxic drug that is fused to) that relates to this treatment of needs.Because the CD200 on the antibody recognition cancer cells, so any this type of cell of described antibodies is destroyed.When independent use antibody killed or removes cancer cells, this type of killed or removes and can realize by starting endogenous host immune function such as CDC and/or ADCC.Measure antibody whether by this way the assay method of cell killing in those skilled in the art's limit of power.

Therefore, in one embodiment, antibody of the present disclosure can be used to send the various kinds of cell toxic chemical.Any cytotoxic compound can be fused to this antibody.Can realize merging by chemistry or genetics (for example, by developed by molecule) method as single fusion.Cytotoxic compound can be biological, as polypeptide, and perhaps small molecules.As the skilled person will appreciate, for small molecules, use chemistry to merge, and, can use chemistry or heredity to merge for biological compound.

The limiting examples of cytotoxic compound comprises molecule, bioprotein and its mixture of compound, plant, fungi or the bacterial origin of curative drug, emitted radiation.Cytotoxic drug can be the cytotoxic drug of effect in the cell,, for example comprises short distance, high-energy alpha emitter as the short-range radiation radiator.Enzyme activity toxin and its segmental example are diphtheria toxin A fragments, the non-binding active fragments of diphtheria toxin, exotoxin A (from Pseudomonas aeruginosa (Pseudomonasaeruginosa)), ricin A chain, abrin A chain, mould lotus root toxalbumin IIA chain, α-sacrin, some tung oil tree (Aleurites fordii) albumen, some Dianthus caryophyllus L. toxalbumin, dyers' grapes (Phytolacca americana) albumen ((PAP, PAPII and PAP-S), balsam pear (Morodica charantia) inhibitor, curcin, crotin, Saponaria officinalis (Saponaria officinalis) inhibitor, spend more white tree toxalbumin, mitogillin, restrictocin, phenomycin and enomycin.The method of the enzymatic activity polypeptide of preparation immunotoxin is described in WO84/03508 and WO85/03508, and they are incorporated herein by reference.Some cytotoxicities part is from for example, Zorubicin, Chlorambucil, daunomycin, methotrexate, neocarzinostatin and platinum.

The method of puting together antibody with cytotoxic agent had been described and in the past within those skilled in the art's limit of power.

Alternatively, antibody coupling can be arrived the energy-rich radiation radiator, for example, radio isotope, as ¹³¹I, gamma emitter, it causes killing of some cell dias when being positioned at tumor locus.For example see, S.E.Order, ＂ Analysis, Results, and Future Prospective of theTherapeutic Use of Radiolabeled Antibody in Cancer Therapy ＂, Monoclonal Antibodies for Cancer Detection and Therapy, people such as R.W.Baldwin (eds.), pp 303-316 (Academic Press 1985) is introduced into this paper as a reference.Other suitable radio isotope comprise alpha emitter, as ²¹²Bi, ²¹³Bi and ²¹¹At and beta emitter, as ¹⁸⁶Re and ⁹⁰Y.

In some embodiments, this CD200 binding antibody provides immunosuppressant benefit among the blocking-up CLL by directly deciding the leukemia cell through the CD200 target.Especially, stimulating immune system can allow to remove the CLL cell from spleen and lymphoglandula.The applicant does not know that also deciding B cell activity agent (as A Lun pearl monoclonal antibody) with target simply realizes successfully removing the CLL cell from these microenvironments.On the contrary, the CLL reaction-ive T cell can be than antibody better near these organs.In other embodiments, by having realized that with anti-CD200 labeling of monoclonal antibodies CLL cell direct cell kills.

According to composition of the present disclosure and method, in the embodiment that is particularly useful, directly cell kills the especially effective means that cancer therapy is provided to the combination of the driving of Th1 spectrum with immunne response.Thereby, in one embodiment, cancer therapy is provided, and wherein to cancer patients's administration of antibodies or antibody fragment, described antibody or antibody fragment are in conjunction with CD200 and a) interaction and the b between blocking-up CD200 and its acceptor) directly kill the cancer cells of expressing CD200.The mechanism of kill cancer cell can include but not limited to ADCC and/or CDC; Merge with toxin; Merge with radio-labeling; With the biological agents that relates to cell killing such as granzyme B or perforin fusion; Merge with cytotoxicity virus; With cytokine such as TNF-α or IFN-α fusion.In alternative embodiment, cancer therapy relates to administration of antibodies, and this antibody a) is blocked interaction and b between CD200 and its acceptor) strengthen cytotoxic T cell or NK cytoactive to tumour.The enhancing of this type of cytotoxic T cell or NK cytoactive can be for example be combined by antibody and cytokine such as IL-2, IL-12, IL-18, IL-13 and IL-5 are merged.In addition, can realize this type of enhancing by anti-CD200 antibody and inhibitor such as ImiDs, thalidomide or thalidomide analogue combined administration.

In a further embodiment, cancer therapy relates to administration of antibodies, the interaction between this antibody (1) blocking-up blocking-up CD200 and its acceptor and (2) with the T cytotaxis to tumour cell.By being merged, Ab and chemokine such as MIG, IP-10, I-TAC, CCL21, CCL5 or LIGHT can realize the T cytotaxis.And, can cause the desirable rise of LIGHT with the chemotherapeutic treatment.Blocking immunity suppress and by antibody target surely directly the compound action of kill tumor cell provide the peculiar methods of enhanced effect.

Also can be used as diagnostic tool according to anti-CD200 antibody of the present disclosure.Can express at the CD200 of treatment Pretesting examination of living tissue or cancer cell tissue sample so as prediction separately or with the effect of the anti-CD200 therapy of other promoting agents or method combination (as chemotherapeutic, radiotherapy, immunomodulatory therapy or the like).For example, use the blood that obtains from the patient who suffers from the hematopoiesis cancer, use anti-CD200 antibody and suitable cancer cells marker such as CD38 and CD19 on the CLL cell combined, can assess the expression of CD200 on the cancer cells by facs analysis.Can select the CD200 level to be used for anti-CD200 Antybody therapy than the patient of at least 1.4 times of the level height of finding on the normal B cell.As another example, can be with anti-CD200 Antybody therapy from the expression of patient's tissue sample with CD200 in the pernicious and normal cell of measuring the patient.

In another example that uses anti-CD200 antibody as diagnosis or prognosis instrument, obtain suffering from malignant tumour the patient biopsy samples and use anti-CD200 antibody by FACS or use anti-CD200 to measure the expression of CD200 by immunohistochemistry.If compare with corresponding healthy tissues, at least 1.4 times of higher levels of CD200 of tumor cells expression select the cancer patients to be used for immunomodulatory therapy (including but not limited to comprise the therapy of anti-CD200 therapy) so.For from not expressing the cancer of the cell of CD200 usually, detectable CD200 shows the potentially useful of anti-CD200 therapy on the cancer biopsy samples.The immunomodulatory therapy can be anti-CD200 therapy, but also can be to influence immune any other therapy of patient.The promoting agent that the example of suitable immunomodulatory therapy comprises the negative adjusting of using blocking t cell or antigen presenting cell (for example, anti-CTLA 4, anti--PD-L1, anti--PDL-2, anti--PD-1) or use the promoting agent (for example, anti-CD40 or anti-4-1BB) of the positive common stimulation that strengthens the T cell.In addition, the immunomodulatory therapy can be a cancer vaccine, as irregular peptide or tumour cell peptide, the dendritic cell that it produces cytotoxic T cell or loads tumour cell, perhaps use increase NK cell number or T cytoactive promoting agent (for example, anti-CD200 cell makes up separately or with inhibitor such as ImiDs, thalidomide or thalidomide analogue), perhaps use to exhaust and (for example regulate the agent of T cell activity, anti-CD200 antibody separately or with the ONTAK combination), perhaps Plasmacytoid dendritic cell.It also is favourable making up with the promoting agent that increases the migration of T cell or dendritic cell, as any promoting agent of blocking-up SPARC.In addition, the immunomodulatory therapy can be a cancer vaccine, as the heat shock protein(HSP) that loads the exosome tumour RNA in dendritic cell, patient source of tumour cell or tumour DNA, oncoprotein or tumour peptide, patient source (hsp ' s), loads the hsp ' s of tumour antigen or stimulates adjuvant the tumour system at different levels, any other compound as the acceptor (for example, toll sample acceptor) of CpG, Luivac, Biostim, Ribominyl, Imudon, Bronchovaxom or activation innate immune system.And therapy can comprise with cytokine such as IL-2, GM-CSF and IFN-γ treatment.The also combination of the impaired active promoting agent (as map kinase inhibitor) of dendritic cell in imagination and the recovery tumor environment.

In one embodiment, this antibody also can be used for detecting in vivo cancerous cells.By traget antibody, will be applied to the experimenter through the antibody of mark, then experimenter's imaging is realized detecting in the body.According to the example that can be used for the mark of diagnosing image of the present disclosure be radio-labeling as ¹³¹I, ¹¹¹In, ¹²³I, ^99mTc, ³²P, ¹²⁵I, ³H, ¹⁴C and ¹⁸⁸Rh, fluorescent mark, isotropic substance, the chemiluminescent substance of the emission positron that can detect as fluorescein and rhodamine, nucleus magnetic resonance activity mark, by positron emission tomography (＂ PET ＂) scanner are as luciferin, and enzyme labelling, as peroxidase or Phosphoric acid esterase.Also can use short distance radiation radiator, as passing through short distance detector probe such as the detectable isotropic substance of transrectal probe.Use technology known in the art, can be with this type of reagent traget antibody.About relating to the radiolabeled technology of antibody, for example see, see Wensel and Meares, Radioimmunoimaging and Radioimmunotherapy, Elsevier, N.Y. (1983) is introduced into this paper as a reference.Also see people such as D.Colcher, ＂ Use of MonoclonalAntibodies as Radiopharmaceuticals for the Localization of HumanCarcinoma Xenografts in Athymic Mice ＂, Meth.Enzymol.121:802-816 (1986) is introduced into this paper as a reference.

Can be used for the in-vitro diagnosis test according to radiolabeled antibody of the present disclosure.The specific activity of antibody, its bound fraction, probe or part depends on how radiolabeled half life, isotopic purity and mark are incorporated in the biological reagent.In immunoassay test, specific activity is high more usually, and sensitivity is good more.Method with labelled with radioisotope antibody is well known in the art.

Radiolabeled antibody can be applied to the patient, wherein it navigates to and has the antigenic cancer cells that this antibody reacts with it, and uses known technology such as radioactive nuleus to scan, and uses for example γZhao Xiangji or emission tomography photograph detection or " imaging ".For example see, people such as Bradwell, ＂ Developmentsin Antibody Imaging ＂, Monoclonal Antibodies for Cancer Detection andTherapy, people such as R.W.Baldwin, (eds.), pp.65-85 (Academic Press1985) is introduced into this paper as a reference.Alternatively, can be with positron radiation X tomography scanner (as be positioned at Brookhaven National Laboratory the positron radiation X tomography scanner that is called Pet VI), wherein radio-labeled emission positron is (for example, ¹¹C, ¹⁸F, ¹⁵O and ¹³N).

The biological reagent that can partly prepare fluorophore and chromophore's mark from standard known in the art.Because antibody and other protein adsorption wavelength are up to the light of about 310nm, so should select the fluorescence part to have the substance absorption at about 310nm and the wavelength that preferably is higher than 400nm.Multiple suitable fluorescent agent and chromophore be by Stryer, Science, and 162:526 (1968) and Brand, people such as L., Annual Review of Biochemistry, 41:843-868 (1972) describes, and is introduced into this paper as a reference.Can be by ordinary method such as U.S. Patent number 3,940,475,4,289,747 and 4,376, the method described in 110 (being introduced into this paper as a reference) fluorescence chromophore moieties traget antibody.

In according to another embodiment of the present disclosure, the progress of monitor therapy treatment and/or the method for validity are provided.This method relates to be used immunomodulatory therapy and at least twice and measures among the experimenter CD200 level to determine the validity of this therapy.For example, level before the treatment of CD200 can be determined, and after using at least therapy, the CD200 level can be measured once more.The reduction of CD200 level shows effective treatment.The doctor can be used as the guidance that increases dosage or therapy frequency with the measurement of CD200 level.Certainly should be appreciated that and directly to monitor the CD200 level, perhaps alternatively, can monitor any marker relevant with CD200.The additive method of determining the validity of this therapy includes but not limited to detect cancer cells, total lymphocyte counting, lymphoglandula size, regulatory T cells number, serum cytokines spectrum, perhaps T or the secretion B cell or the cell within a cell factor or cytokine as measuring by ELISPOT.

C. other CD200 antagonists

Be used for that the CD200 antagonist of the disclosure and polypeptide and/or antibody are adapted to diagnosis as described herein especially and treatment is used.Therefore, CD200 antagonist and anti-CD200 antibody and its variant can be used for therapy, comprise combination treatment, are used for the diagnosis and the prognosis of disease, and the monitoring that is used for progression of disease.

In treatment embodiment of the present disclosure, the expection bi-specific antibody.Bi-specific antibody is monoclonal, preferred people or humanized antibody, and it has binding specificity at least two kinds of different antigens.In this case, one of binding specificity is that pair cell (as cancer cells or immunocyte) is gone up the antigenic binding specificity of CD200, and another is to arbitrary other antigens, the binding specificity of preferred pair cell surface protein or acceptor or receptor subunit.

The method for preparing bi-specific antibody is in those skilled in the art's scope.Routinely, the reorganization of bi-specific antibody produces and is based on two coexpressions that heavy chain immunoglobulin/light chain is right, and two heavy chains have different specificity (Milstein and Cuello, Nature, 305:537-539 (1983)).Antibody variable territory with binding specificity (antibody-antigen combination site) of hope can be merged with immunoglobulin (Ig) constant domain sequence.Merge preferably and merge with the heavy chain immunoglobulin constant domain, described heavy chain immunoglobulin constant domain comprises to small part hinge, CH2 and CH3 district.If coding heavy chain immunoglobulin fusions and hope, the DNA of light chain immunoglobulin can be inserted in the independent expression vector, and cotransfection is in the appropriate host biology.For the further details of the current known property the illustrated method that is used to produce bi-specific antibody, for example see people such as Suresh, Methods in Enzymology, 121:210 (1986); WO 96/27011; People such as Brennan, Science 229:81 (1985); People such as Shalaby, J.Exp.Med.175:217-225 (1992); People such as Kostelny, J.Immunol.148 (5): 1547-1553 (1992); People such as Hollinger, Proc.Natl.Acad.Sci.USA 90:6444-6448 (1993); With people such as Gruber, J.Immunol.152:5368 (1994); With people such as Tutt, J.Immunol.147:60 (1991).Bi-specific antibody also comprise crosslinked or different puting together (heteroconjugate) antibody.Can use the cross-linking method of any routine to prepare the different antibody of puting together.Suitable crosslinking agent is well known in the art, and at U.S. Patent number 4,676, in 980 along with many crosslinking technologicals are open together.

Preparation and the multiple technologies of directly separating bispecific antibody fragment from the reconstitution cell culture have also been described.For example, used leucine zipper to produce bi-specific antibody.People such as Kostelny, J.Immunol., 148 (5): 1547-1553 (1992).Can be connected to the Fab ' part of two kinds of different antibodies by gene fusion from Fos and the proteic leucine zipper peptide of Jun.Can hinge area also the original antibody homodimer form the antibody heterodimer to form monomer and then oxidation.This method also can be used to produce the antibody homodimer.People such as Hollinger, Proc.Natl.Acad.Sci.USA, " double antibody " technology that 90:6444-6448 (1993) describes provides the alternate mechanism of preparation bispecific antibody fragment.Described fragment comprises by joint and is connected to light chain variable structural domain (V _L) weight chain variable structural domain (V _H), described joint is too short and make and match between two structural domains on same the chain.Therefore, segmental V _HAnd V _LStructural domain is compelled to and another segmental complementary V _LAnd V _HThe structural domain pairing, thus two antigen binding sites formed.Also reported by using strand Fv (scFv) dimer to prepare another strategy of bispecific antibody fragment.See people such as Gruber, J.Immunol., 152:5368 (1994).Alternatively, antibody can be described in people Protein Eng.8 (10): 1057-1062 (1995) such as Zapata " linear antibody ".In brief, these antibody comprise the Fd section (V of pair of series _H-C _H1-V _H-C _H1), it forms a pair of antigen binding domain.Linear antibody can be dual specific or monospecific.

D. method of application and preparation

The antibody route of administration of antibody of the present disclosure (no matter be pure antibody, through the antibody of mark, be fused to antibody of toxin or the like) be according to known method, for example, by in intravenously, intraperitoneal, the brain, intramuscular, subcutaneous, intraocular, intra-arterial, intrathecal injection or perfusion, suck or the interior approach of damage, perhaps pass through slow-released system.Preferably by perfusion or bolus injection continuous administration antibody.Can be with part or systemic fashion administration of antibodies.

Can be mixed with antibody of the present invention with pharmaceutically acceptable carrier.The technology that is used to prepare and use disclosure compound can be seen ＂ Remington ' s Pharmaceutical Sciences, ＂ Mack PublishingCo., Easton, PA, latest edition.Can intravenously or by nose or lung, preferably use this therapeutic composition as liquid or powdery aerosol (freeze dried).Parenteral or subcutaneous administration composition as desired.When systemic administration, therapeutic composition should be aseptic, and essentially no pyrogeneous substance and in the acceptable solution of parenteral has due consideration to pH, isotonicity and stability.For example, the essentially no pyrogeneous substance material of pharmaceutical preparation is used as human therapeutic agent so that be suitable for.These conditions are well known by persons skilled in the art.

The pharmaceutical composition that is suitable for using comprises such composition, and wherein one or more antibody of the present invention comprise with significant quantity in order to realize their its intended purposes.More particularly, treat the antibody amount that significant quantity refers to effective prevention, alleviates or alleviates disease symptoms or prolongs the experimenter's who is treated survival.The treatment significant quantity fixes in those skilled in the art's limit of power really, especially according to provided herein open in detail.Can determine to treat effective dose by using in the external and body method.

Although top open, in some embodiments, can use according to the disclosure from the polypeptide of this antibody-like at antibody.

Example

Mouse model and CD200+ cell construction

The Raji/PBL model

With containing 4x10 ⁶200 μ l RPMI of RAJI cell (ATCC) are with 0,1,5 or 1,000 ten thousand PBL subcutaneous injection NOD.CB17-Prkdc＜scid〉mouse (Jackson Laboratory).Every group comprises 9 or 10 mouse.On the histopaque gradient, then use 0.9% ammonium chloride to carry out erythrocyte splitting from 250ml separation of whole blood PBL.Inferior on every Wendesdays monitoring tumor growth with kind of calliper length and width.Calculate gross tumor volume based on length x width x width/2.

By two tails in pairs Si Shi t check analysis with the group of PBL injection with only accept difference between the group of tumour cell.Since the 32nd day, in the group of accepting 5 or 1,000 ten thousand PBL, still in the group of accepting 100 ten thousand PBL, do not observe significant difference.

Namalwa PBL model

With containing 4x10 ⁶200 μ l RPMI of individual Namalwa cell (ATCC) and 0,2 or 1,000 ten thousand PBL subcutaneous injection NOD.CB17-Prkdc＜scid〉and mouse (Jackson Laboratory, BarHarbor, Maine).Every group comprises 9-10 mouse.On the histopaque gradient, then use 0.9% ammonium chloride to carry out erythrocyte splitting from 250ml separation of whole blood PBL.Inferior on every Wendesdays monitoring tumor growth with kind of calliper length and width.Calculate gross tumor volume based on length x width x width/2.

Produce the clone of stable expression CD200

(Invitrogen, Carlsbad CA) produce Raji and the Namalwa clone of stable expression CD200 to use Virapower slow virus expression system (Lentiviral Expression System).Use forward primer 5 '-GACAAGCTTGCAAGGATGGAGAGGCTGGTGA-3 ' (SEQ ID NO:34) and reverse primer 5 '-GACGGATCCGCCCCTTTTCCTCCTGCTTTTCTC-3 ' (SEQ ID NO:35) by RT-PCR from former generation CLL cellular segregation CD200 cDNA.The PCR product cloning is entered carrier pCR8/GW/TOPO-TA and to each cloning and sequencing to Gateway.(CA) clone that will have a correct sequence recombinates among lentiviral vectors pLenti6/V5/DEST and the pLenti6/UbC/V5/DEST so that justice and antisense orientation to be arranged for Invitrogen, Carlsbad to use the Gateway technology.Main difference between these two carriers is to be used to drive CD200 expression promoter: pLenti6/V5/DEST to contain people CMV immediate early promoter, and pLenti6/UbC/V5/DEST contains people's ubiquitin C promotor.

The transient cotransfection of recommending according to the manufacturer that passes through the 293-FT cell produces the false type slow virus of the high VSV-G that tires original seed.By with 10 ⁶Individual cell is resuspended in and contains 12 μ g/ml 1, and 5-dimethyl-1 is in the 1ml growth medium of the poly-Methobromide of 5-phenodiazine 11 methylene radical and add 1ml slow virus original seed transduction Raji or Namalwa cell.37 ℃ spend the night the incubation cell after, remove the substratum that contains virus and replace with the 4ml fresh culture.Two days later, the CD200 by the flow cytometry cells infected expresses.In all experiments, 〉=70% cell is CD200 ⁺, and in parental cell line and the cell with the transduction of negative control (antisense CD200) virus, detect less than CD200.

In order to separate the cloned cell line of expressing CD200, selected the cell that infects 13 days with blasticidin.The concentration of the blasticidin that uses is 6 μ g/ml for the Raji cell, is 2 μ g/ml for the Namalwa cell.Then by with blasticidin resistant cell limiting dilution separating stable clone in 96 orifice plates.Use the mouse anti human CD200 that PE-puts together (clone MRC OX104, Serotec) and the BD FACSCalibur of equipment high-throughput sampler by flow cytometry with 96 well format screening and cloning.Screening is altogether behind 2000 Raji and 2000 the Namalwa clones, will have those clonal expansions that the highest CD200 expresses and be used for further characterizing with routine techniques.

Embodiment 1

The effect of the humanization form of C2aB7 in the RAJI_CD200/PBL model

A) whether in vivo keep their effect in the tumor model for the C2aB7 that assesses the humanization form, in the RAJI-CD200/PBL model, tested chimeric C2aB7 (seeing U.S. Patent Application Publication No. 2005/0129690) and 3 kinds of humanization forms (C2aB7V4V1, C2aB7V3V1 and C2aB7V3V2) and negative control antibody alxn4100.To be subcutaneously injected into NOD.CB17-Prkdc＜scid with the RAJI cell of CD200 transduction〉in the mouse, and being evaluated at chimeric or humanization C2aB7 antibody or control antibodies alxn4100 (its debond tumour cell) exist or do not exist down, PBL reduces the ability of tumor growth.Point out the antibody and the tumour cell of concentration below the initial application, twice intravenously used weekly then.Every group of group below 10 mouse foundation:

Group 1:4 x 10 ⁶RAJI_CD200 is subcutaneous

Group 2:4 x 10 ⁶RAJI_CD200 is subcutaneous+6 x 10 ⁶PBL

Group 3:4 x 10 ⁶RAJI_CD200 is subcutaneous+6 x 10 ⁶PBL+5mg/kg C2aB7

Group 4:4 x 10 ⁶RAJI_CD200 is subcutaneous+6 x 10 ⁶PBL+20mg/kg C2aB7V4V1

Group 5:4 x 10 ⁶RAJI_CD200 is subcutaneous+6 x 10 ⁶PBL+5mg/kg C2aB7V4V1

Group 6:4 x 10 ⁶RAJI_CD200 is subcutaneous+6 x 10 ⁶PBL+20mg/kg C2aB7V3V1

Group 7:4 x 10 ⁶RAJI_CD200 is subcutaneous+6 x 10 ⁶PBL+5mg/kg C2aB7V3V1

Group 8:4 x 10 ⁶RAJI_CD200 is subcutaneous+6 x 10 ⁶PBL+5mg/kg C2aB7V3V2

Group 9:4 x 10 ⁶RAJI_CD200 is subcutaneous+6 x 10 ⁶PBL+20mg/kg alxn4100

Measure length of tumor and width weekly 3 times, and calculate gross tumor volume by length of tumor * width * width/2.Figure 18 shows that the expression of CD200 has stoped immunocyte to reduce gross tumor volume on the tumour cell as expection.The C2aB7 of all humanization forms blocks tumor growth up to 97% under the dosage of 20mg/kg.Control antibodies alxn4100 does not influence tumor growth.These data show that everyone source antibody all is highly effective in the blocking-up tumor growth.

B) by the CD200 immune evasion

Although the human immunity system can cause immunne response to many cancer types, this replys the cancer that is not enough to eliminate in the most of patients, may be the immune evasion that immune negative adjusting is caused owing to by tumour.Among the patient (n=80) that we have identified in all inspections on chronic lymphocytic leukemia (CLL) cell immunosuppression molecule CD200 raised 1.5-5.4 doubly.The interaction of known CD200 and its acceptor changes to Th2 with the cytokine spectrum from Th1 in mixed lymphocyte reacion, and causes inducing of regulatory T cells, thinks that regulatory T cells hinders the immunity of tumour-specific effector T cell.In this research, whether we have handled on the tumour cell expression of CD200 works in immune evasion, thereby whether anti-CD200 Antybody therapy influences tumor growth in this model to the removing of tumour cell with antagonism to stop immunity system in xenotransplantation hu/SCID mouse model.

Be subcutaneously injected in the NOD/SCID mouse with people's non-Hodgkin lymphoma clone RAJI and Namalwa personnel selection CD200 transduction and with human peripheral lymphocyte (PBMC).To accept express in the mouse of tumour growth in time and the tumour cell of accepting not express CD200 in the mouse of tumour cell of CD200 tumour growth phase in time relatively.In experiment subsequently, treat mouse with chimeric or Humanized anti-CD 20 0 antibody (dosage range 1mg/kg is to 20mg/kg) by intravenous injection.Treatment is beginning immediately behind the tumor cell injection or is beginning after 7 days.

When not existing CD200 to express, PBMC reduces RAJI or Namalwa tumor growth up to 75%.Compare, RAJI or the Namalwa growth of tumor of expressing CD200 on the level suitable with CLL are not reduced by PBMC.In research process, even when behind the tumor injection during 7 days begin treatments, the anti-CD200 antibody of using 5mg/kg causes almost completely that tumor growth suppresses (little tumour has taken place 1/10 mouse).

The existence of people CD200 has suppressed the ability of human lymphocyte elimination tumour cell on the tumour cell.Express with the anti-CD200 Antybody therapy of antagonism CD200 tumor suppression tumor growth, show the potentiality of anti-CD200 therapy as the method likely of CLL.

C) C2aB7G1 is to the effect of C2aB7G2/G4 construct

Whether the same with the G1 construct effective or more effective in order to assess the anti-CD200 antibody that do not have effector function (as the G2/G4 fusion constructs of the C2aB7 that describes below), G1 and G2/G4 form and the humanization form (alxn5200) of test C2aB7 in the Raji_CD200/PBL model.Above-mentioned RAJI cell with CD200 transduction is subcutaneously injected into NOD.CB17-Prkdc＜scid〉in the mouse, and assessed inosculating antibody CD200 antibody c2aB7Gl (c2aB7), c2aB7G2/G4 or humanization form hC2aB7V3V1G1 (V3V1) or hC2aB7V3V2G1 (V3V2) control antibodies alxn4100 exists or not in the presence of, PBL reduces the ability of tumor growth.The following antibody of concentration of pointing out is used with tumour cell at first, then intravenously administered twice weekly.Every group of group below 10 mouse foundation:

Group 1:4 x 10 ⁶RAJI_CD200 is subcutaneous

Group 2:4 x 10 ⁶RAJI_CD200 is subcutaneous+6 x 10 ⁶PBL

Group 3:4 x 10 ⁶RAJI_CD200 is subcutaneous+6 x 10 ⁶PBL+20mg/kg hV3V2-G1

Group 4:4 x 10 ⁶RAJI_CD200 is subcutaneous+6 x 10 ⁶PBL+5mg/kg alxn 5200

Group 5:4 x 10 ⁶RAJI_CD200 is subcutaneous+6 x 10 ⁶PBL+2.5mg/kg alxn 5200

Group 6:4 x 10 ⁶RAJI_CD200 is subcutaneous+6 x 10 ⁶PBL+1mg/kg alxn 5200

Group 7:4 x 10 ⁶RAJI_CD200 is subcutaneous+6 x 10 ⁶PBL+20mg/kgchC2aB7G2/G4

Group 8:4 x 10 ⁶RAJI_CD200 is subcutaneous+6 x 10 ⁶PBL+5mg/kgchC2aB7G2/G4

Group 9:4 x 10 ⁶RAJI_CD200 is subcutaneous+6 x 10 ⁶PBL+2.5mg/kgchC2aB7G2/G4

Group 10:4 x 10 ⁶RAJI_CD200 is subcutaneous+6 x 10 ⁶PBL+1mg/kgchC2aB7G2/G4

Group 11:4 x 10 ⁶RAJI_CD200 is subcutaneous+6 x 10 ⁶PBL+20mg/kg alxn 4100

Measure length of tumor and width weekly three times, and calculate gross tumor volume by length of tumor * width * width/2.Figure 19 shows that as expected, the CD200 on the tumour cell expresses and stops immunocyte to reduce tumor growth.Yet, add anti-CD200 antibody and reduce gross tumor volume up to 100%.Although 20mg/kg C2aB7Gl causes 6/10 medium and small tumor growth of mouse, with 1 mouse growth tumour only in the group of 20mg/kgC2aB7G2/G4 treatment, prompting G2/G4 form may cause than G1 form better or the effect that equates at least.All anti-CD200 antibody comprise the humanization form, all block tumor growth under 5mg/kg fully.Do not reduce tumor growth with the control antibodies treatment.The G2/G4 form of these digital proofs C2aB7 is highly effective in the tumor growth of blocking-up expression CD200.These data prove that further it is highly effective that the humanization form of C2aB7 is blocked in the tumor growth in this model.

D) generation of G2/G4 construct

Change plasmid with two steps, at first with in the people CH1 district from Age I site by terminator codon to the IgG1 district replacing that is positioned at the BamH I site behind the SV40 poly a-signal.Digest C2aB7-6 and cC7 G2G4 (L-SIGN antibody) with Age I and BamHI, and handle C2aB7-6 with CIP.By electrophoresis and gel extraction purifying from 10 of C2AB7-6,315bp fragment and from the 1752bp fragment of cC7G2G4.These fragments are connected, and electroporation and is seeded on the LB/carb/gluc flat board in the XL1Blue intestinal bacteria.Also use Qiagen miniprep post DNA isolation at the growth from solution bacterium colony.Opposite with the IgGl fragment, determine the segmental existence of IgG2G4 Age I/BamHI by Pvu II digestion, described digestion causes existing 267 and two bands of 1152bp, and is opposite with the band of 1419bp.Select clone 21 to be used for further use.

By produce the remainder in CH1 district with the IgG2/G4 form with overlapping PCR from the variable region end to Age I site.Containing the CH1 district begins to produce in the production of plasmid cC7G2G4 before the PCR fragment in Age I site.Primer C7mhHF (TCCTCAGCCTCCACCAAGGGCC, SEQ ID NO:1) and Rev Age Pri (GGGCGCCTGAGTTCCACGAC, SEQ ID NO:2) are used for the PCR reaction, and G2G4 63L1D is used as template to produce the 142bp fragment.Use primer C2aB7 rev (GGCCCTTGGTGGAGGCTGAGGAAACTGTGAGAGTGGTGC, SEQID NO:3) and lacpri (GCTCCCGGCTCGTATGTTGTGT, SEQ ID NO:4), Fab C2aB7 is produced mouse variable region of heavy chain (with the upstream material) as template with the fragment of about 1250bp.These fragments by electrophoresis and gel extraction purifying and with primer Rev Age Pri (GGGCGCCTGAGTTCCACGAC, SEQ ID NO:2) and LeadVHpAX (ATATGAAATATCTGCTGCCGACCG, SEQ ID NO:5) is used for the fragment that overlapping PCR produces 558bp, with its purifying on the PCR purification column.This 558bp fragment is digested to produce the 458bp fragment, with this fragment electrophoresis and gel extraction purifying with clone 21 usefulness Xho I and Age I.Also will clone 21 usefulness Xho I and Age I digestion, handle with CIP, by electrophoresis and gel extraction purifying 11.6kb fragment.Connect these fragments and electroporation in the XL1Blue intestinal bacteria and be seeded on the LB/carb/gluc flat board.Find when digesting that clone C2aB7G2G4.11 has the restriction fragment of expection with Pvu II.

To final construct C2AB7G2G4.11 order-checking.The TAA terminator codon of discovery light chain has been mutated into sequence TCA and has made and will add 6 extra amino acid at the C-terminal of light chain.There is C2AB7-6 in discovery in the clone of IgG1 form, it is the parent of C2AB7G2G4.11.The antibody that contains the G2G4 construct is described in Figure 10,11,12,13 and 15.

Embodiment 2

CD200 on the cancer cells expresses

A. measure CD200 rise among the CLL patient

Anti-CD200 (Serotec) dyeing that the anti-CD5 (e-bioscience) that will put together with FITC from 15 CLL patients' lymphocyte, the anti-CD19 (e-bioscience) that APC puts together and PE put together.Correspondingly to lymphocyte dyeing from healthy donor.The CD200 that measures on the CD5+CD19+ cell expresses.As shown in Figure 20, although the CD200 expression level is different in CLL patient's sample, all CLL samples demonstrate the level (1.6-4 times of scope) of rising, compare with the CD200 expression on the normal B cell to have higher CD200 expression.The CLL patient that selection demonstrates the CD200 expression of elevated levels is used for carrying out anti-CD200 treatment according to methods described herein.

B. to the facs analysis of cancerous cell line

Use one group of NCI60 clone to express by facs analysis assessment CD200, described clone is from melanoma patient, patients with prostate cancer, glioblastoma patient, astrocytoma patient, neuroblastoma patient, ovarian cancer patients, patients with lung cancer and patients with renal cell carcinoma.According to manufacturer's specification sheets (Invitrogen) Zenon-Alexa488 mark C2aB7.500,000 to 100 ten thousand cells with the antibody staining of 1 μ g mark 20 minutes, are then washed with PBS.With FACSCalibur (BectonDickinson) assessment cell dyeing.With the dyeing of the cell of antibody labeling with keep unlabelled sample relatively and determine dyeing/undyed ratio.In Figure 21, greater than 1 but less than 2 ratio be pointed out as+/-, the ratio between 2 to 3 is+, between 2 to 10 be ++,〉10 ratio be +++.For the clone of glioblastoma, astrocytoma, prostate cancer or lung cancer test is not expressed CD200, and do not list below.4,2/2 ovarian cancer cell line in the melanoma cell series of 5 tests, 2/3 kidney cell line, 2/2 neuroblastoma clone and 1/3 breast cancer cell tie up to cell surface with can detected horizontal expression CD200, and the prompting solid tumor also can use CD200 as immune evasion mechanism.

C. to the RT-QPCR of patient's sample

In order to verify whether CD200 is not only raised on clone, and on former generation patient sample, also raised, carried out RT-QPCR and immunohistochemistry (IHC) with former generation patient sample.Obtain RNA sample from Cytomix from ovarian cancer and melanoma patient.Preparation cDNA also uses the 10ng/ml yeast rna with 1:100 and 1:1000 dilute sample.The CD200 that provides with ABI measures Hs00245978_ml, moves QPCR with sample.For 18S normalization method, in order to 1:10, the sample operation 18S of 000 dilution measures (ABI).Each diluent is with duplicate operation.Ovarian cancer and melanoma patient sample and CLL patient's sample to 18S normalization method, are determined to express with respect to the multiple of normal PBL then.

Figure 22 has shown that the CD200 on the ovarian cancer patients sample expresses.Slurries/slurries transfer/as if the corpora mammillaria slurry samples has the highest CD200 to be expressed, than high about 10 to 20 times of normal PBL.CD200 is expressed in uterine endometrium, mucus and the clear-cells sample relatively low, all or subnormal ovary expression level (doubly) than the high 1-5 of normal PBC.

Figure 23 has shown that some melanoma shift sample: the CD200 expression level of jejunum, small intestine, lymphoglandula, lung, skin and brain.Some of these samples are normal/tumours of coupling, point out by numeral (1 pair to-2 pairs).There are not other extra samples of the normal specimens of coupling to be used for comparison by operation yet.The jejunum sample demonstrates than the remarkable higher CD200 expression level of normal organ, shifts sample than the high about 4-7 of normal jejunum doubly.

D. to the immunohistochemistry of former generation patient sample

Carry out IHC with 2 refrigerated melanoma patient samples (LifeSpan).D1B5 and C2aB7Fab fragment are used for dyeing.IgG1 antibody is as the isotype contrast.Detect the combination of one-level antibody with anti-mouse secondary antibody and DAB chromophoric group.

As shown in Figure 24, two kinds of melanoma samples of test all demonstrate the strong film dyeing with anti-CD200 antibody, but do not dye with the isotype contrast.Normal skin tissue does not demonstrate CD200 dyeing.These data show that CD200 not only fastens at melanoma and ovarian cancer cell and are raised, and raised on former generation patient sample.

E. melanoma and ovarian tumor cell raise by immunosuppression molecule CD200 and carry out immune evasion

Immune evasion is the key feature of cancer progression.Tumour can be escaped immunity system by multiple mechanism, and every kind of mechanism all is the important obstruction of immunotherapy.Realize that new and the immunotherapy of effective form more will need to understand these processes and their similaritys in cancer with different.We had identified the immunosuppression molecule CD200 that will be raised in the past on the chronic lymphocytic leukemia cell.The existence of CD200 is reduced effective cytotoxic T cell and is replied required Th1 cytokine generation.We illustrate human tumor cells in animal model CD200 expresses prevention human lymphocyte repulsion tumour, and has suppressed tumor growth with the anti-CD200 Antybody therapy of antagonism.In this research, we assessed CD200 whether raise be found on other cancers and these cancer cells on CD200 express whether influence immunne response.

By RT-QPCR quantitative adenocarcinoma ovaries (slurries/slurries shift/the nipple slurries, uterine endometrium, mucus, clear-cells) and malignant melanoma transfer patient sample in relative CD200 information level.

Assessed with normal skin by IHC and to have compared the cell surface expression of CD200 in two melanoma and three ovarian cancers (slurries) patient freezing tissue sample with normal ovarian.Use the anti-CD200 antibody of PE mark, express by CD200 on the cell surface of facs analysis assessment melanoma cell series SK-MEL-5, SK-MEL-24 and SK-MEL-28 and ovarian cancer cell line OV-CAR-3.The cancerous cell line influence that the pair cell factor is composed in mixed lymphocyte reacion of CD200 is expressed in assessment in dendritic cell by cell being added person monocytic cell source and allotypic human T-cell's the culture.The cytokine that detects in the supernatant liquor by ELISA produces (for Th1 is IL-2 and IFN-γ, is IL4 and IL10 for Th2).

Quantitative PCR shows 20 times up to normal PBL of in slurries adenocarcinoma ovaries sample CD200 expression level, and equals or up to 4 times of normal ovarian.In uterus the CD200 in film, mucus and the clear-cells adenocarcinoma ovaries sample is expressed as or is lower than the normal ovarian level.As if in transferring to the malignant melanoma metastatic tumor of jejunum, the CD200 expression level is significantly higher than normal specimens.In the malignant melanoma lung shifted, 2/6 demonstrates the CD200 higher than normal specimens expressed.

IHC is presented at intensive on the malignant cell of two melanoma patients, CD200 dyeing that specific membranes is relevant.The normal skin sample demonstrates the faint dyeing of endotheliocyte.In three ovarian cancer patients, one demonstrates intensive CD200 dyeing, a medium positive by name, a subclass that demonstrates faint painted tumour cell.In all three kinds of situations, matrix all demonstrates strong dyeing.

CD200 melanoma cell series SK-MEL-24 and SK-MEL-28 and on the cell surface of ovarian cancer cell line OV-CAR-3 high level expression, and medium level is expressed on melanoma cell series SK-MEL-5.In mixed lymphocyte reacion, add any downward modulation Th1 production of cytokines of these clones, and the clone of not expressing CD200 does not cut the Th1 production of cytokines, shows direct dependency.Eliminated this effect comprising of the anti-CD200 antibody of antagonism in the training period.

Melanoma and ovarian tumor cell can raise CD200, thereby may suppress efficient immune.Can allow immunity system to cause the effective cytotoxic response of antitumor cell with the anti-CD200 treatment of antagonism.

F. express of the influence of the cancerous cell line of CD200 to cytokine spectrum in the mixed lymphocyte reacion

In mixed lymphocyte reacion, assessed and crossed the cell of expressing CD200 and cytokine response is replied (IL-2, IFN-γ) deflection Th2 from Th1 reply (IL-4, ability IL-10).As the cell source of expressing CD200, the cell that uses the CD200 cells transfected or be from the CD200 positive cancer cell.

Use and use IL-4, GM-CSF and IFN-γ from sophisticated 250,000 dendritic cell of human peripheral mononuclear cell and 1x10 ⁶Individual responsive cell is set up mixed lymphocyte reacion in 24 orifice plates.Responsive cell for can (Ficoll) with phenanthrene from the lymphocyte that is rich in the T cell of peripheral blood purifying.By with peripheral blood cells incubation 1 hour and get non-adherent cell grade separating/enriching T cell in tissue culture flasks.In the anti-CD200 antibody existence of 30 μ g/ml or not, add from 500,000 cells of melanoma cell series SK-MEL-1, SK-MEL-24, SK-MEL-28, ovarian cancer cell line OVCAR3 and non-Hodgkin lymphoma clone Namalwa or former generation CLL cell as positive control to described dendritic cell.Collect the existence of the supernatant liquor and the analysis of cells factor after 48 and 68 hours.

With the cytokine of finding in the ELISA quantitative tissue culture supernatant such as IL-2, IFN-γ and IL-10.From R+D Systems (Minneapolis, MN) obtain every kind of cytokine coupling catch and to detect antibody right, use the recombinant human cytokine to produce the typical curve of every kind of cytokine.The antibacterial agent capture antibody is coated on the flat board with optimum concn in PBS.Spend the night behind the incubation, washing is dull and stereotyped and use the PBS that contains 1%BSA and 5% sucrose to seal 1 hour.After the PBS washing that contains 0.05%Tween 3 times, supernatant liquor adds with twice or 10 times of diluents of the PBS that contains 1%BSA.Then detect captive cytokine with suitable biotinylated anti-cytokine antibodies by streptavidin and the SigmaS substrate that the adding alkaline phosphatase is puted together.With the dull and stereotyped reader of ELISA (Molecular Devices) assessment colour developing.

As shown in Figure 25 A, the existence with clone (MEL-24, MEL-28, OVCAR-3) of high CD200 expression causes the downward modulation of Th1 cytokine such as IL-2 and IFN-γ.Compare, adding MEL-1 (low CD200 expresses) or Namalwa (no CD200 expresses) does not influence the cytokine spectrum.The anti-CD200 antibody hB7VH3VL2 that adds 50 μ g/ml has recovered Th1 fully and has replied (Figure 25 B), and the anti-CD200 Antybody therapy that shows melanoma or ovarian cancer patients can be that treatment is gone up useful.

Embodiment 3 removes the activated T cell by C2aB7-G1 and its derivative

In the cancer model that uses the tumour cell of not expressing CD200, whether have effect in order to assess anti-CD200 treatment, Namalwa cell and people PBL are expelled in the NOD/SCID mouse, and as following processing mouse.In this model, CD200 only exists only on the immunocyte such as B cell and folliculus t helper cell of natural expression CD200.

The group design:

10 animal/groups

Group 1:4 x 10 ⁶Namalwa is subcutaneous

Group 2:4 x 10 ⁶Namalwa is subcutaneous+8 x 10 ⁶PBL

Group 3:4 x 10 ⁶Namalwa is subcutaneous+8 x 10 ⁶PBL+20mg/kg hV3V2-G1

Group 4:4 x 10 ⁶Namalwa is subcutaneous+8 x 10 ⁶PBL+5mg/kg hV3V2-G1

Group 5:4 x 10 ⁶Namalwa is subcutaneous+8 x 10 ⁶PBL+2.5mg/kg hV3V2-G1

Group 6:4 x 10 ⁶Namalwa is subcutaneous+4 x 10 ⁶PBL

Group 7:4 x 10 ⁶Namalwa is subcutaneous+8 x 10 ⁶PBL+20mg/kg chC2aB7-G2G4

Group 8:4 x 10 ⁶Namalwa is subcutaneous+8 x 10 ⁶PBL+5mg/kg chC2aB7-G2G4

Group 9:4 x 10 ⁶Namalwa is subcutaneous+8 x 10 ⁶PBL+2.5mg/kg chC2aB7-G2G4

Group 10:4 x 10 ⁶Namalwa is subcutaneous+4 x 10 ⁶PBL+20mg/kgchC2aB7-G2G4

Group 11:4 x 10 ⁶Namalwa is subcutaneous+8 x 10 ⁶PBL+20mg/kg alxn4100

In injection mixture, comprise 1/10 dosage.Dosage subsequently is intravenously twice, 3 weeks by a definite date weekly.

Measure length of tumor (L) and width (W) three times weekly and calculate gross tumor volume by L*W*W/2.Figure 26 showed as former foundation, injected people PBL simultaneously and the Namalwa cell has suppressed tumor growth.Do not observe the influence of chC2aB7-G2G4 to the tumor growth inhibition of PBL mediation.Compare, use the tumor growth inhibition that ALXN5200 (hB7VH3VL2-G1) has blocked the PBL mediation.As if when not having CD200 on the tumour cell, during the anti-CD200 Antybody therapy that carries out with the antibody of mediation effector function such as G1 construct, the effector cell of key is eliminated in the PBL colony.These Notes of Key Datas do not have the antibody of effector function to compare with use, and when use had the antibody of effector function, the validity of anti-CD200 cancer therapy was lower.Yet using the anti-CD200 treatment of the construct with effector function is useful in (as transplanting in background or the autoimmune disease) under the situation of hope removal immunocyte in treatment.

Embodiment 4

T cell killing by hB7VH3VL2

Whether cause killing and wounding of T cell in order to assess the activated T cell with anti-CD200 antibody (for example G1) incubation that contains the constant region that mediates effector function, T cell activation and following foundation are killed and wounded mensuration.

A) CD3+T cellular segregation

Use Accuspin ^TMSystem, the density gradient centrifugation by the heparinization whole blood obtains human peripheral lymphocyte (PBL) from the normal health volunteer.To each Accuspin pipe (Sigma, St.Louis, MO; Cat# A2055) adds 15ml Histopaque-1077 (Sigma, St.Louis, MO; Cat#H8889), then with described pipe with 1500rpm (rev/min) make to allow Histopaque pass frit in centrifugal 2 minutes.The 30ml whole blood is at the frit higher slice and do not having under the arresting situation under the room temperature with 2000rpm centrifuge tube 15 minutes.Collect the PBL interface and with centrifugal 10 minutes of 1200rpm with having 2% heat-inactivated foetal calf serum (FBS) (Atlas Biologicals, Ft.Collins, CO; Cat# F-0500-D) twice of PBS washing monocyte.According to manufacturer's specification sheets by passing HTCC-5 post (R﹠amp; D Systems) separation of C D3+T cell.The cell of washing wash-out, counting, and be resuspended among the RPMI 1640 that contains 5% heat-inactivated single donor's serum, 2mM L-glutaminate, 10nM Hepes and penicillin/streptomycin.

B. with dull and stereotyped bonded mOKT3 activation

Spend the night the incubation bag by the hole of 12 orifice plates (Falcon) by being used in the 10 μ g/mL mOKT3 (Orthoclone) that dilute among the PBS at 4 ℃.Remove residual antibody and use the gentle washing plate of PBS.Will be as the CD3+T cell of above-mentioned isolating purifying with 2x10 ⁶Among the RPMI1640 that contains 5% heat-inactivated single donor's serum, 2mM L-glutaminate, 10nM Hepes and penicillin/streptomycin in the final concentration adding flat board in/hole.Cell is containing 5%CO ₂Humidified incubator in 37 ℃ kept 72 hours down.

C.mOKT3 activatory CD3+ target cell ⁵¹The chromium mark

When incubation period finishes, results mOKT3 activatory CD3+ cell, washing and with 10 ⁷Individual cell/mL is resuspended among the RPMI1640 that does not have serum.By adding 125 μ Ci ⁵¹Chromium (PerkinElmer, Billerica, MA)/10 ⁶Individual cell came chromaking (chromate) cell in following 2 hours at 37 ℃.Results are through the cell of mark, washing and with 2x10 in the RPMI that contains 5% heat-inactivated single donor's serum ⁵The final concentration of individual cell/mL is resuspended in the identical substratum.

D. from body NK effector cell's preparation

By density gradient centrifugation such as the above-mentioned human peripheral lymphocyte (PBL) that obtains from same individual.Collect the PBL interface and with centrifugal 10 minutes of 1200rpm with having 2% heat-inactivated foetal calf serum (FBS) (Atlas Biologicals, Ft.Collins, CO; Cat# F-0500-D) PBS washing monocyte.Specification sheets according to the manufacturer is gone up the positive separation of C D56+ cell of selecting by the magnetic bead of puting together at anti-CD56 (MiltenyiBiotec, Auburn, CA, Cat# 120-000-307).The cell of washing wash-out, counting, and with 1.3x10 ⁶Individual cell/mL is resuspended among the RPMI1640 that contains 5% heat-inactivated single donor's serum, 2mM L-glutaminate, 10nM Hepes and penicillin/streptomycin.Containing 5%CO ₂Humidified incubator under 37 ℃ with 4x10 ⁶The final concentration of individual cells/well is the incubated overnight cell in the 3mL same medium.When incubation period finishes, harvested cell, washing is counted and is resuspended in and contains 2mM L-glutaminate, 10mM Hepes, 2x10 ^-5Among the serum-free RPMI of M 2 mercapto ethanol and penicillin/streptomycin.

The E.ADCC assay method

Will be as the mOKT3 activatory CD3+ target cell of the 51Cr mark of above-mentioned preparation with 10 ⁴Individual cells/well is distributed in the hole of 96 orifice plates with 50 μ L.Results CD56+ effector cell, the washing, the counting and with 2.5x10 ⁶Individual cell/mL (make the effector cell: target cell is than being 25:1) or 10 ⁶Individual cell/mL (make the effector cell: target cell is than being 10:1) resuspension and distribution (100 μ L/ hole) are in the hole of containing target cell.10 times of diluents that will resist CD200 antibody (V3V2-G1 or V3V2-G2/G4) join effector cell and target cell with the final concentration of 10,1,0.1 and 0.01 μ g/mL.Measuring contrast comprises following: effector cell and target cell (0Ab) when 1) not having antibody; Target cell when 2) not having the effector cell (spontaneous cracking) and 3) with the effector cell of 0.2% Tween-80 incubation and target cell (maximum release).Cultivate to carry out all cells in triplicate.Containing 5%CO ₂Humidified incubator in 37 ℃ of following culturing cells 4 hours.When incubation period finished, centrifugal flat board was transferred in the scintillation vial and with γ scintillometer (Wallac) with sedimentation cell and with 150 μ L cell conditioned medium liquid and is counted.According to following formula the result is expressed as special cracking per-cent:

(running sample counting (the cpm)-average spontaneous cracking of per minute)/(average maximum cracking-average spontaneous cracking) * 100.

F. flow cytometry

To be assigned to 96 hole circle base plates (Falcon, Franklin Lakes NJ as the 100 μ l cell suspending liquids (the CD56+NK cell of mOKT3 activatory CD3+ cell or purifying) of above-mentioned preparation; Cat#353077) in the hole.With being combined in 4 ℃ of incubation cells shown in the following material 30 minutes: fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, the antibody of PerCP-Cy5.5-or allophycocyanin (APC)-put together is (all from Becton-Dickinson, San Jose, CA); Anti-people CD25-FITC (cat#555431); Anti-people CD3-APC (cat# 555335); Anti-humen CD 20 0-PE (cat# 552475); Anti-people CD8-PerCP-Cy5.5 (cat# 341051); Anti-people CD4-APC (cat# 555349); Anti-people CD5-APC (cat# 555355) and anti-people CD56-APC (cat# 341025).Also comprise the isotype contrast of every kind of traget antibody.Behind FACS damping fluid washed cell twice (centrifugal 3 minutes of 1800rpm), cell is resuspended in 300 μ l PBS (Mediatech, Herndon, VA; Cat#21-031-CV) in and use FacsCaliber instrument and CellQuest software (Becton Dickinson, San Jose CA) pass through flow cytometry.

As shown in figure 27, the activated T cell demonstrates the high CD200 expression on they surfaces.When the NK cell was used as the effector cell, the activated T cell was effectively killed (Figure 28) in the presence of VH3VL2-G1 rather than VH3VL2-G2G4.These data show that the anti-CD200 antibody with effector function can eliminate the activated T cell.This antibody can have therepic use in the treatment of transplanting background or autoimmune disease.

Except regulatory T cells, shown that the Plasmacytoid dendritic cell have negative immunoregulation effect (Wei S in human cancer, Kryczek I, Zou L, Daniel B, Cheng P, MottramP, Curiel T, Lange A, Zou W Plasmacytoid dendritic cells induce CD8+regulatory T cells in human ovarian carcinoma.Cancer Res.2005 Jun 15; 65 (12): 5020-6).Therefore, it can be favourable eliminating the therapy of Plasmacytoid dendritic cell and the combination of anti-CD200 therapy.

Embodiment 5

CD200 on the plasmocyte

At first by using the medullary cell of ammonium chloride cracking erythrocyte preparation from 10 multiple myeloma patients and 3 normal donors.Cell is resuspended in the FACS damping fluid and with following mixtures of antibodies mark:

●K-FITC/CD38-PE/CD138-PerCP-Cy5.5

●λ-FITC/CD38-PE/CD138-PerCP-Cy5.5

● isotype contrast-FITC/CD38-PE

●CD200-FITC/CD38-PE

Use BD FACS Canto to collect data also with BD DiVA software analysis.Analyze the expression that the bright cell (plasmocyte) of CD38 is gone up CD200.As shown in figure 29, a part of plasmocyte is expressed CD200 with high strength in normal donor.In multiple myeloma patients, most plasmocyte are expressed CD200.

In the multiple myeloma background, be similar to CLL or other cancers of expressing CD200, the CD200 of tumour cell expresses and can stop immunity system to remove tumour cell.The anti-CD200 therapy of antagonism can allow immunity system to remove cancer cells subsequently.The fixed anti-CD200 therapy of plasmacytic ablative of target can have the treatment benefit in autoimmunization or transplanting background.

Embodiment 6

CD200 on the virus

CD200 also expresses on many viruses such as myxoma virus M141R or human herpesvirus 8,hhv 8.The CD200 that is similar on tumour cell expresses, and the CD200 on the virus can stop immunity system effectively to remove virus.In the infection of the virus of expressing CD200, have the treatment benefit with the anti-CD200 Antybody therapy of antagonism, allow immunity system to remove virus.Alternatively, can use the anti-CD200 antibody of ablative.

To understand and to make multiple modification to embodiment disclosed herein.For example, as it will be understood by those skilled in the art that particular sequence disclosed herein to change a little and not necessarily unfriendly influence be used for polypeptide, antibody or antibody fragment functional in conjunction with OX-2/CD200.For example, can in the antibody sequence of being everlasting, carry out one or more amino acid replacements and not destroy antibody or segmental functional.Thereby should be appreciated that with specific antibodies as herein described has greater than the polypeptide of 70% identity degree or antibody in the scope of the present disclosure.In the embodiment that is particularly useful, imagination and specific antibodies as herein described have the antibody greater than about 80% identity.In other useful embodiments, imagination and specific antibodies as herein described have the antibody greater than about 90% identity.Therefore, top description should not be understood that to limit, and only is the example as preferred embodiment.Those skilled in the art will imagine other modifications in the scope of the present disclosure and spirit.

Reference

Following reference is incorporated herein more completely to describe the state in field under the present invention.Those publications that these following publications or top reference are introduced and any inconsistent will the solution between the disclosure to help mode of the present disclosure.

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Claims

1. the anti-CD200 antibody or its Fab that comprise the constant region of change, wherein said antibody or Fab demonstrate the effector function that weakens with respect to unmanifest anti-CD200 antibody.

2. the antibody of claim 1 or Fab, the effector function that wherein weakens comprise following one or more:

Compare with unmanifest anti-CD200 antibody

A. the cytotoxicity (ADCC) of the dependence antibody of Jiang Diing;

B. the cytotoxicity (CDC) of the dependence complement of Jiang Diing.

3. the antibody of claim 1 or Fab, wherein said antibody is murine antibody, chimeric antibody, humanized antibody, single-chain antibody, perhaps people's antibody.

4. the antibody of claim 1 or Fab, wherein said antibody or its Fab are selected from IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgA, IgD and IgE.

5. the antibody of claim 1 or Fab, wherein said constant region has been engineered to and has comprised at least one amino acid replacement, insertion or disappearance.

6. the antibody of claim 1 or Fab, it comprises the aminoacid sequence of nucleic acid encoding, described nucleic acid under stringent condition be selected from SEQ ID NOS:12,14,16,17 and 21 nucleotide sequence or the hybridization of its fragment.

7. the antibody of claim 1 or Fab, it comprises the aminoacid sequence that at least 90% identity is arranged with SEQ ID NOS:13,15,18 or 22 aminoacid sequence or its fragment.

8. the antibody of claim 1 or Fab, wherein said constant region comprises the feature below one or more:

I) glycosylation of Gai Bianing;

Ii) Ala-Ala sudden change;

Iii) be selected from SEQ ID NOS:13,15,18 and 22 G2/G4 construct.

9. the antibody of claim 8, the glycosylation of wherein said change comprise following one or more: (i) change in one or more sugar components; The (ii) existence of one or more sugar components; The (iii) disappearance of sugar component.

10. the antibody of claim 9, wherein said antibody is expressed in the host cell that is selected from mammalian cell, bacterial cell and vegetable cell.

11. the antibody of claim 10, wherein said host cell is intestinal bacteria.

12. the antibody of claim 10, wherein said host cell is rat-hybridoma.

13. the antibody of claim 10, wherein said host cell is a Chinese hamster ovary celI.

14. the antibody of claim 1 or Fab, it comprises the feature below one or more:

With respect to unmanifest anti-CD200 antibody

A) to the combination that reduces of one or more Fc acceptors;

B) the ADCC activity that reduces; With

C) the CDC activity that reduces.

15. the antibody of claim 1 or Fab, the wherein said antibody anti-CD200 antibody that is closure.

16. the antibody of claim 15 or Fab, wherein said antibody are murine antibody, chimeric antibody, humanized antibody, single-chain antibody or people's antibody.

17. comprise antibody or its Fab of one or more aminoacid sequences, described one or more aminoacid sequences are by nucleic acid encoding, described nucleic acid is hybridized with the nucleotide sequence that is selected from SEQ IDNOS:10 and 25 or its fragment under stringent condition.

18. comprise antibody or its Fab of one or more aminoacid sequences, described one or more aminoacid sequences have at least 90% identity with the fragment in amino acid 20 beginnings that is selected from SEQ ID NO:11, SEQ ID NO:26, SEQ ID NO:11, SEQ ID NO:26's in the fragment of amino acid 23 beginnings and other segmental aminoacid sequences of SEQ ID NOS:11 and 26.

19. comprise antibody or its Fab of one or more aminoacid sequences, described one or more aminoacid sequences are by nucleic acid encoding, described nucleic acid is hybridized with the nucleotide sequence that is selected from SEQ IDNOS:10 and 27 or its fragment under stringent condition.

20. comprise antibody or its Fab of one or more aminoacid sequences, described one or more aminoacid sequences have at least 90% identity with the fragment in amino acid 20 beginnings that is selected from SEQ ID NO:11, SEQ ID NO:28, SEQ ID NO:11, SEQ ID NO:28's in the fragment of amino acid 23 beginnings and other segmental aminoacid sequences of SEQ ID NOS:11 and 28.

21. comprise antibody or its Fab of one or more aminoacid sequences, described one or more aminoacid sequences are by nucleic acid encoding, described nucleic acid is hybridized with the nucleotide sequence that is selected from SEQ IDNOS:8 and 25 or its fragment under stringent condition.

22. comprise antibody or its Fab of one or more aminoacid sequences, described one or more aminoacid sequences have at least 90% identity with the fragment in amino acid 20 beginnings that is selected from SEQ ID NO:9, SEQ ID NO:26, SEQ ID NO:9, SEQ ID NO:26's in the fragment of amino acid 23 beginnings and other segmental aminoacid sequences of SEQ ID NOS:9 and 26.

23. comprise antibody or its Fab of one or more aminoacid sequences, described one or more aminoacid sequences are by nucleic acid encoding, described nucleic acid is hybridized with the nucleotide sequence that is selected from SEQ IDNOS:12 and 27 or its fragment under stringent condition.

24. comprise antibody or its Fab of one or more aminoacid sequences, described one or more aminoacid sequences have at least 90% identity with the fragment in amino acid 20 beginnings that is selected from SEQ ID NO:13, SEQ ID NO:28, SEQ ID NO:13, SEQ ID NO:28's in the fragment of amino acid 23 beginnings and other segmental aminoacid sequences of SEQ ID NOS:13 and 28.

25. comprise antibody or its Fab of one or more aminoacid sequences, described one or more aminoacid sequences are by nucleic acid encoding, described nucleic acid is hybridized with the nucleotide sequence that is selected from SEQ IDNOS:14 and 23 or its fragment under stringent condition.

26. comprise antibody or its Fab of aminoacid sequence, described aminoacid sequence has at least 90% identity with the fragment in amino acid 21 beginnings that is selected from SEQ ID NO:15, SEQ ID NO:24, SEQ ID NO:15, SEQ ID NO:24's in the fragment of amino acid 21 beginnings and other segmental aminoacid sequences of SEQ ID NOS:15 and 24.

27. comprise antibody or its Fab of one or more aminoacid sequences, described one or more aminoacid sequences are by nucleic acid encoding, described nucleic acid is hybridized with the nucleotide sequence that is selected from SEQ IDNOS:16 and 27 or its fragment under stringent condition.

28. comprise antibody or its Fab of one or more aminoacid sequences, described one or more aminoacid sequences have at least 90% identity with the fragment in amino acid 20 beginnings that is selected from SEQ ID NO:13, SEQ ID NO:28, SEQ ID NO:13, SEQ ID NO:28's in the fragment of amino acid 23 beginnings and other segmental aminoacid sequences of SEQ ID NOS:13 and 28.

29. comprise antibody or its Fab of one or more aminoacid sequences, described one or more aminoacid sequences are by nucleic acid encoding, described nucleic acid is hybridized with the nucleotide sequence that is selected from SEQ IDNOS:17 and 29 or its fragment under stringent condition.

30. comprise antibody or its Fab of one or more aminoacid sequences, described one or more aminoacid sequences have at least 90% identity with the fragment in amino acid 21 beginnings that is selected from SEQ ID NO:18, SEQ ID NO:30, SEQ ID NO:18, SEQ ID NO:30's in the fragment of amino acid 21 beginnings and other segmental aminoacid sequences of SEQ ID NOS:18 and 30.

31. comprise antibody or its Fab of one or more aminoacid sequences, described one or more aminoacid sequences are by nucleic acid encoding, described nucleic acid is hybridized with the nucleotide sequence that is selected from SEQ IDNOS:19 and 31 or its fragment under stringent condition.

32. comprise antibody or its Fab of one or more aminoacid sequences, described one or more aminoacid sequences have at least 90% identity with the fragment in amino acid 21 beginnings that is selected from SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:20, SEQ ID NO:32's in the fragment of amino acid 21 beginnings and other segmental aminoacid sequences of SEQ ID NOS:20 and 32.

33. comprise antibody or its Fab of one or more aminoacid sequences, described one or more aminoacid sequences are by nucleic acid encoding, described nucleic acid is hybridized with the nucleotide sequence that is selected from SEQ IDNOS:21 and 33 or its fragment under stringent condition.

34. comprise antibody or its Fab of one or more aminoacid sequences, described one or more aminoacid sequences have at least 90% identity with the fragment in amino acid 20 beginnings that is selected from SEQ ID NO:22, SEQ ID NO:34, SEQ ID NO:22, SEQ ID NO:34's in the fragment of amino acid 20 beginnings and other segmental aminoacid sequences of SEQ ID NOS:22 and 33.

35. each Fab of claim 17-34, wherein said Fab demonstrates the effector function that weakens.

36. each antibody or Fab of claim 1 and 17-34, wherein said antibody or Fab are conjugated to material.

37. the antibody of claim 36 or Fab, wherein said material is selected from toxin, enzyme, therapeutical agent, diagnostic reagent and developer.

38. anti-CD200 antigen-binding fragments of antibodies, wherein said Fab are demonstrated the half life of prolongation in the experimenter by modification.

39. the Fab of claim 38, wherein said Fab comprises the feature below one or more:

A) be added into polyoxyethylene glycol;

B) be fused to another polypeptide; With

C) be connected to small molecules.

40. the Fab of claim 39, wherein said another polypeptide is in conjunction with serum protein.

41. the Fab of claim 39, wherein said another polypeptide is an albumin.

42. the Fab of claim 39, wherein said small molecules is in conjunction with serum protein.

43. claim 38 and 39 each Fabs, wherein said Fab is a single-chain fragment.

44. reduce CD200 positive cell number purpose method among the patient, it comprises uses anti-CD200 antibody or its Fab to described patient.

45. the method for claim 44, wherein said antibody or Fab suppress the interaction of CD200 and its acceptor.

46. suppress the interactional method of CD200 and its acceptor, it comprises administration of anti-cd 20 0 antagonist.

47. the method for claim 46, wherein said CD200 antagonist reduces the expression of CD200.

48. the method for claim 46, wherein said antagonist are selected from polypeptide, small molecules, chemical, metal, organometallic compound, mineral compound, nucleic acid, oligonucleotide, aptamers, spiegelmer, immunomodulator, Fab, prodrug and peptide simulated compound.

49. the method for claim 47, wherein said antagonist is selected from double-stranded DNA, single stranded DNA, double-stranded RNA, single stranded RNA, RNAi and antisense nucleic acid.

50. the method for claim 46, wherein said antagonist is in conjunction with the CD200 acceptor.

51. the method for claim 46, wherein said antagonist reduces the CD200 receptor expression.

52. the method for claim 50, wherein said antagonist are selected from polypeptide, small molecules, chemical, metal, organometallic compound, mineral compound, nucleic acid, oligonucleotide, aptamers, immunomodulator, Fab, prodrug and peptide simulated compound.

53. the method for claim 51, wherein said antagonist is selected from double-stranded DNA, single stranded DNA, double-stranded RNA, single stranded RNA.

54. the method for claim 46, wherein said antagonist are the anti-CD200 antibody that demonstrates the effector function that weakens.

55. the method for claim 54, wherein said anti-CD200 antibody demonstrate the T cell ability extremely of reduction.

56. the method for claim 46, wherein said anti-CD200 antibody is blocking antibody.

57. treatment cancer patients's method, it comprises that wherein said antagonist is selected from polypeptide, small molecules, chemical, metal, organometallic compound, mineral compound, nucleic acid, oligonucleotide, aptamers, immunomodulator, Fab, prodrug and peptide simulated compound to described patient's administration of anti-cd 20 0 antagonist.

58. treatment cancer patients's method, described method comprises antibody or the Fab of described patient being used claim 1.

59. the method for claim 58, wherein said cancer is from the neural crest cell cancer.

60. the method for claim 58, wherein said cancer is selected from plasmocyte cancer, ovarian cancer, skin carcinoma, lung cancer, kidney, mammary cancer, prostate cancer, neuroblastoma, lymphoma, myelomatosis and leukemia.

61. treatment cancer patients's method, described method comprise described patient is used each antibody or Fab of claim 17-34.

62. treatment method for cancer, described method comprise the anti-CD200 antibody of combined administration or its fragment and second kind of promoting agent or therapy.

63. the method for claim 62, wherein said second kind of promoting agent comprises the feature below one or more:

A) chemotherapy activity;

B) to the adjusting activity of T cell; With

C) immunoregulatory activity.

64. the method for claim 62, wherein said second kind of promoting agent or therapy are selected from radiotherapy, chemotherapeutic, immunomodulator, irregular (heteroclitic) peptide, antibody, Fab, nucleic acid, small molecules, organometallic compound, polypeptide, aptamers, spiegelmer, chemical, mineral compound, metal, prodrug and peptide simulated compound.

65. the treatment method for cancer, it comprises uses each anti-CD200 antibody or its fragment of claim 1-5.

66. the method for claim 65, it also comprises uses the active promoting agent of the dendritic cell that can recover impaired in the tumor environment.

67. the method for claim 66, wherein said promoting agent is a map kinase inhibitor.

68. the method for claim 65, it also comprises uses chemotherapeutic.

69. the method for claim 68, wherein said chemotherapeutic is selected from aminoglutethimide, amsacrine, Anastrozole, asparaginase, bcg, bicalutamide, bleomycin, buserelin, busulfan, camptothecine, capecitabine, carboplatin, carmustine, Chlorambucil, cis-platinum, CldAdo, clodronate disodium, colchicine, endoxan, cyproterone, cytosine arabinoside, Dacarbazine, gengshengmeisu, daunorubicin, Dienestrol, stilboestrol, docetaxel, Zorubicin, pidorubicin, estradiol, estramustine, Etoposide, Exemestane, filgrastim, fludarabine, fluohydrocortisone, Fluracil, Fluoxymesterone, Flutan, gemcitabine, genistein, Coserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, Interferon, rabbit, irinotecan, letrozole, folinic acid, leuprorelin acetate, LEVAMISOLE HCL, chlorethyl cyclohexyl nitrosourea, mustargen, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, Ortho-para-prism DDD, mitoxantrone, Nilutamide, the Luo Keda azoles, Sostatin, oxaliplatin, taxol, Pamidronate, pentostatin, Plicamycin, porfimer sodium, procarbazine, Raltitrexed, sharp appropriate uncommon agate, U-9889, Suramine, tamoxifen, Temozolomide, teniposide, testosterone, Tioguanine, thio-tepa, titanocene dichloride, topotecan, Si Tumanbu, tretinoin, vinealeucoblastine(VLB), vincristine(VCR), vindesine and vinorelbine.

70. the method for claim 68, it also comprises uses antiangiogenic agent.

71. the method for claim 68, wherein said chemotherapeutic is a metabolic antagonist.

72. the method for claim 71, wherein said metabolic antagonist is a pyrimidine analogue.

73. the method for claim 72, wherein said pyrimidine analogue is selected from 5 FU 5 fluorouracil, floxuridine, capecitabine, gemcitabine and cytosine arabinoside.

74. the method for claim 71, wherein said metabolic antagonist is a purine analogue.

75. the method for claim 74, wherein said purine analogue is selected from mercaptopurine, Tioguanine, pentostatin and 2-chlorodeoxyadenosine.

76. the method for claim 68, it also comprises uses antimitotic agent, microtubule disrupting agent, dna damage agent, microbiotic, anti-platelet agents, DNA alkylating agent, anticoagulant, fibrinolytic agent, anti-migration agent, secretion inhibitor agent, immunosuppressor, immunomodulator, growth factor receptor inhibitors, topoisomerase enzyme inhibitor, reflunomide and chromatin disrupting agent.

77. the method for claim 76, wherein said immunosuppressor are selected from S-Neoral, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine and mycophenlate mofetil.

78. the method for claim 76, wherein said immunomodulator are selected from thalidomide and its analogue.

79. the method for claim 76, wherein said immunomodulator is selected from Revlimid, Actimid and endoxan.

80. the method for claim 76, wherein said immunomodulator is selected from irregular peptide and cancer vaccine.

81. the method for claim 68, wherein order or use described second kind of promoting agent simultaneously.

82. the method for virus infection among the treatment patient, it comprises uses anti-CD200 antibody or its Fab to described patient.

83. the method for claim 82, wherein said antibody or its Fab are closure.

84. the method for claim 82, wherein said antibody or its Fab are each antibody or its Fabs of claim 1-5.

85. comprise anti-CD200 antibody or its Fab of the constant region that makes a variation, wherein said antibody is compared with unmanifest anti-CD200 antibody and is demonstrated the enhanced effector function.

86. the antibody of claim 85 or its Fab, wherein said antibody are murine antibody, chimeric antibody, humanized antibody, single-chain antibody, perhaps people's antibody.

87. the antibody of claim 85 or Fab, wherein said antibody is selected from IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgA, IgD and IgE.

88. the antibody of claim 85 or Fab, wherein said antibody comprises the feature below one or more:

Compare with unmanifest antibody

A) to the combination of the increase of one or more Fc acceptors;

B) the ADCC activity of Zeng Jiaing; With

C) the CDC activity of Zeng Jiaing.

89. the antibody of claim 85 or Fab, wherein said constant region comprises the glycosylation of change.

90. the antibody of claim 85 or Fab, wherein said constant region comprise at least one aminoacid insertion, disappearance or substitute.

91. the antibody of claim 89, the glycosylation of wherein said change comprise following one or more: (i) change of one or more sugar components; The (ii) existence of one or more sugar components; The (iii) disappearance of sugar component.

92. the antibody of claim 91, wherein said antibody is expressed in the host cell that is selected from mammalian cell, bacterial cell and vegetable cell.

93. the antibody of claim 92, wherein said host cell is intestinal bacteria.

94. the antibody of claim 92, wherein said host cell is rat-hybridoma.

95. the antibody of claim 92, wherein said host cell is a Chinese hamster ovary celI.

96. the antibody of claim 85 or Fab, wherein said antibody or its Fab are the anti-CD200 antibody of closure or its Fab.

97. the antibody of claim 96 or its Fab, wherein said antibody are chimeric antibody, humanized antibody or people's antibody.

98. the antibody of claim 85 or its Fab, wherein said antibody or its Fab are the anti-CD200 antibody of misclosure or its Fab.

99. the antibody of claim 98 or its Fab, wherein said antibody are chimeric antibody, humanized antibody, single-chain antibody or people's antibody.

100. the method for claim 44, wherein said CD200 positive cell is selected from B cell and T cell.

The method of claim 44, it comprises antibody or the Fab of using claim 85.

The method of claim 44, wherein said patient suffers from autoimmune disease.

The method of claim 102, wherein said autoimmune disease is selected from rheumatoid arthritis, inflammatory bowel, systemic lupus erythematous, multiple sclerosis, Hashimoto thyroiditis, pernicious anemia, Addison disease, type i diabetes, dermatomyositis, Sjogren syndrome, lupus erythematosus, myasthenia gravis, reiter syndrome and Graves' disease.

The method of claim 44, wherein said patient accepts maybe will accept graft.

The method of claim 104, wherein said patient has accepted maybe will accept allograft.

The method of claim 44, wherein said antibody or Fab are misclosures.

The method of claim 44, wherein said patient is the women.

The method of claim 107, wherein before using described antibody or its fragment to described patient screening pregnant state.

The method of claim 44, wherein before using the therapy that comprises described antibody or its Fab to cell or tissue sample test CD200 expression level from described patient.

110. suppress the material of CD200 and its acceptor interaction, wherein said material does not cause effector function.

111. the material of claim 110, wherein said material is selected from: aptamers, polypeptide, immunomodulator, Fab, small molecules, chemical, organometallic compound, mineral compound, metal, nucleic acid, oligonucleotide, prodrug and peptide simulated compound.

112. comprise pharmaceutical composition in conjunction with the material of CD200.

113. the pharmaceutical composition of claim 112, wherein said material are anti-CD200 antibody or its Fab.

114. the pharmaceutical composition of claim 113, wherein said antibody or its Fab are each antibody or Fabs of claim 1-34,38-43,85-99.

115. claim 112 or 113 each pharmaceutical compositions, its essentially no pyrogeneous substance.

116. the pharmaceutical composition of claim 114, its essentially no pyrogeneous substance.

117. the method for monitoring therapy progress, it comprises from the expression of accepting or plan is accepted the patient collection organization sample or the cell at least twice of described therapy and measured CD200 on the sample of described collection.

118. the method for claim 117, wherein said patient suffers from cancer.

119. the method for claim 117, wherein the method below one or more is measured the CD200 expression:

(a) immunohistochemistry,

(b) flow cytometry.

120. the method for claim 117 is wherein collected at least a described sample before the described therapy of beginning.

121. claim 44,58-60,83,103,104,106,117 each methods, wherein said patient did not accept the radiotherapy of brain.

122. claim 44,58-60,83,103,104,106,117 each methods, wherein said patient did not accept the brain operation in the past.

123. comprise the anti-CD200 antibody or its Fab that go immunity of the constant region of change, wherein said antibody or Fab demonstrate the effector function that weakens with respect to unmanifest anti-CD200 antibody.

124. comprise the anti-CD200 antibody or its Fab that go immunity of the constant region of variation, the wherein said antibody of immunity or its Fab of going compared with unmanifest anti-CD200 antibody and demonstrated the enhanced effector function.

125. claim 123 or 124 go the immunity antibody or Fab, wherein said go the immunity antibody or Fab be the anti-CD200 antibody of closure or its Fab.

126. claim 124 go the immunity antibody or Fab, wherein said go the immunity antibody or Fab be the anti-CD200 antibody of misclosure or its Fab.