CN101443040A - Methods of treating lupus using CD4 antibodies - Google Patents

Abstract

Methods of treating lupus, including systemic lupus erythematosus, cutaneous lupus erythmetosus, and lupus nephritis, are provided. The methods involve administration of a combination of a non-depleting CD4 antibody and another compound used clinically or experimentally to treat lupus. Methods of treating lupus nephritis by administration of a non-depleting CD4 antibody that results in an improvement in renal function and/or a reduction in proteinuria or active urinary sediment are also provided. Methods of treating multiple sclerosis by administration of a non-depleting CD4 antibody, optionally in combination with another compound used clinically or experimentally to treat MS, are described. Methods of treating transplant recipients and subjects with rheumatoid arthritis, asthma, psoriasis, Crohn's disease, ulcerative colitis, and Sjogren's syndrome are also provided.

Description

Method with CD4 Antybody therapy lupus

The cross reference of related application

[0001] the application is the non-provisional utility patent application of the priority of temporary patent application and rights and interests formerly below the requirement: USSN 60/783535, Bryan Irving submitted on March 16th, 2006, be entitled as " METHODS OF TREATING LUPUS USING CD4 ANTIBODIES " (with the method for CD4 Antybody therapy lupus), with USSN 60/873881, Bryan Irving submitted in December in 2006 on the 7th, be entitled as " METHODS OF TREATING LUPUS USING CD4 ANTIBODIES " (with the method for CD4 Antybody therapy lupus), for all purposes, they are included in separately by reference in full.

Invention field

[0002] the present invention relates to individually or be used in combination non-expendable (non-depleting) CD4 antibody with other chemical compounds and treat the lupus of mammalian object and the method for other autoimmune conditions.

Background of invention

[0003] autoimmune disease still is human important clinical problems as systemic lupus erythematosus (sle) (SLE), myasthenia gravis, multiple sclerosis and idiopathic thrombocytopenic purpura etc.As its name suggests, autoimmune disease causes its considerable damage by the immune system of health self.Although the pathology of various types of autoimmune diseasees mechanism is different, total mechanism relates to combining of some antibody (being called autoreactivity antibody or autoantibody in the literary composition) of existing among the patients serum and self nuclear antigen or cellular antigens.

[0004] lupus is a kind of autoimmune disease that relates to the antibody of attacking connective tissue.Suffering from this disease in respect of 1,000,000 Americans in advance, wherein mainly is the 20-40 women in year.The principal mode of lupus is a systemic lupus (systemic lupus erythematosus (sle); SLE).The generation of SLE and antinuclear antibody, circulating immune complex and the activation of complement system are relevant.Per 700 20-60 between year the women have 1 people that SLE takes place approximately.SLE can influence all tracts and can cause serious tissue injury.There are many not homospecific autoantibodys among the SLE.The autoantibody that SLE patient produces has Anti-DNA, anti--Ro and anti--blood-platelet specific usually and can cause the Clinical symptoms that this is sick, and is unusual as glomerulonephritis, arthritis, oromeningitis, neonate CHB and hematology.These autoantibodys also may relate to central nervous system disorder.Arbuckle etc. have described SLE clinical episodes generation (Arbuckle etc., (2003) N.Engl.J.Med.349 (16): 1526-1533) of autoantibody before.Exist with double-stranded n DNA and have the diagnostic flag that immunoreactive antibody is used as SLE usually.

[0005] not treat may be fatal to lupus, because its can develop into the internal from attacking skin and joint, comprising lung, heart and kidney (relating generally to nephropathy).Lupus mainly shows as a series of breaking out, and has the interval that the disease performance is light or do not have.Be one of the most serious zone of pathogenicity associated injury among the SLE by the injury of kidney of albuminuretic quantitative determination in the urine, account at least 50% of this disease mortality rate and sickness rate.

[0006] also there is not treatment to diagnose the patient's who suffers from SLE method at present.In fact, the doctor adopts many potent immunosuppressive drugs such as high dose corticosteroids usually, for example, and prednisone or azathioprine or cyclophosphamide, these medicines give during breaking out, but also can experience the patient who frequently breaks out for a long time.Even if effectively treatment has alleviated symptom and prolonged life, but many these medicines have potential serious side effects to the patient who is treated.In addition, these immunosuppressants can disturb and individual produce all antibody and be not the ability of autoreactivity Anti-DNA antibody only.Immunosuppressant can also weaken the ability that health is defendd other potential pathogens, thereby makes the patient to infecting and other potential fatal diseases such as cancer and susceptible thereof.In some these type of situations, the side effect of Therapeutic Method and the lasting low-level outbreak of disease at present can cause serious harm and premature death.

[0007] nearest therapeutic scheme comprises that cyclophosphamide, methotrexate, antimalarial, hormone therapy are (for example, DHEA) and anti--hormonotherapy (for example, anti--prolactin antagonist medicine bromocriptine).The method of the treatment SLE that relates to antibody has also been described.For example, the method (U.S. Patent number 4690905) of Diamond etc. comprises that generation uses these anti--idiotype antibodies to remove the pathogenicity Anti-DNA antibody in the patient system at the monoclonal antibody (this monoclonal antibody is called as anti--idiotype antibody therein) of Anti-DNA antibody then.Yet the time removing a large amount of blood in treatment is a kind of danger and complicated process.U.S. Patent number 6726909 has disclosed the method for treatment SLE, and the Anti-DNA that wherein will comprise purification is anti--and the antibody compositions of idiotype antibody gives the patient, and this administration requires injection or other equivalent mode of administration to carry out.

[0008] high dose intravenous immunoglobulin immunoglobulin (IVIG) infusion also is used to treat some autoimmune disease.So far, SLE provides mixed effect with the IVIG treatment, comprising both treating lupus nephritis (Akashi etc., J.Rheumatology17:375-379 (1990)), and in a few cases, can worsen albuminuria and injury of kidney (Jordan etc., Clin.Immunol.Immunopathol.53:S164-169 (1989)).

[0009] lupus patient, as showing the SLE patient of lupus nephritis clinical evidence and the patients with lupus nephritis needs are economical and safe Therapeutic Method, this method will help to alleviate those tissue injurys that finally cause renal failure and the chronic hemodialysis that these symptoms are caused and/or the demand of renal transplantation.Similarly, other autoimmune diseasees also need effectively and the Therapeutic Method of safety as multiple sclerosis (MS), rheumatoid arthritis, myasthenia gravis, psoriasis, juvenile onset diabetes, sjogren disease, thyroid disease and patients with inflammatory bowel.

Summary of the invention

The conventional embodiment of [0010] one class provides the treatment mammalian object, as the method for the lupus of human subjects.In described method, give described object with the non-expendable CD4 antibody and at least a second combination of compounds of treatment effective dose, described second chemical compound is selected from down group: for example cyclophosphamide, mycophenolate, CTLA4-Ig and α 4-alpha 2 integrin antibodies etc.In some embodiment, described to liking the people.In some embodiment, described second chemical compound is a cyclophosphamide.

[0011] in a class embodiment, described non-expendable CD4 antibody has heavy chain amino acid sequence shown in light-chain amino acid sequence shown in heavy chain amino acid sequence shown in light-chain amino acid sequence shown in heavy chain amino acid sequence, the SEQ ID NO:15 shown in light-chain amino acid sequence shown in heavy chain amino acid sequence, the SEQ ID NO:9 shown in light-chain amino acid sequence shown in the SEQ ID NO:3 and the SEQ ID NO:6 and the SEQ ID NO:12 and the SEQ ID NO:18 or the SEQ ID NO:21 and the SEQ ID NO:24.

[0012] in a class embodiment, described non-expendable CD4 antibody comprises the CD4 binding fragment of the antibody that contains following sequence: heavy chain amino acid sequence shown in light-chain amino acid sequence shown in heavy chain amino acid sequence shown in light-chain amino acid sequence shown in heavy chain amino acid sequence, the SEQ ID NO:15 shown in light-chain amino acid sequence shown in heavy chain amino acid sequence, the SEQ ID NO:9 and the SEQ ID NO:12 and the SEQ ID NO:18 or the SEQ ID NO:21 and the SEQ ID NO:24 shown in light-chain amino acid sequence shown in the SEQ ID NO:3 and the SEQ ID NO:6.

[0013] in a class embodiment, described non-expendable CD4 antibody comprises CDR1 (SEQ ID NO:25), CDR2 (SEQ ID NO:26) or the preferred CDR3 (SEQ ID NO:27) of light chain shown in Figure 1A; For example, this antibody can comprise CDR1, CDR2 and the CDR3 (that is, SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27) of light chain shown in Figure 1A.Similarly, in a class embodiment, described antibody comprises CDR1 (SEQ ID NO:28), CDR2 (SEQ ID NO:29) or the preferred CDR3 (SEQ ID NO:30) of heavy chain shown in Fig. 1 D; For example, this antibody can comprise CDR1, CDR2 and the CDR3 (that is, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30) of heavy chain shown in Fig. 1 D.In one embodiment, described antibody comprises CDR1, CDR2 and the CDR3 (that is SEQ ID NO:25-30) of heavy chain shown in CDR1, the CDR2 of light chain shown in Figure 1A and CDR3 and Fig. 1 D.Other exemplary antibodies include, but not limited to Fig. 1-4 in arbitrary shown in the identical bonded antibody of epi-position of antibody.

[0014] described non-expendable CD4 antibody can be humanized antibody, for example, wherein to treat to as if the people.Described antibody can have nonglycosylated (aglycosylated) Fc part.Randomly, described antibody is not in conjunction with the Fc receptor.In some embodiment, described antibody is the anti--CD4 variant antibody in conjunction with the FcRN receptor.This antibody is chosen wantonly on one or more positions in Fc district amino acid position 270,322,326,327,329,313,333 and/or 334 and is comprised aminoacid replacement, this replacement has changed the Clq combination of antibody and/or has depended on the cytotoxicity (for example, with regard to the parental generation antibody that does not comprise this replacement) of complement.In some embodiment, described antibody comprises remedies receptors bind epi-position or serum albumin binding peptide.Randomly, described antibody comprises three or more antigen binding site.

[0015] normally systemic lupus erythematosus (sle) (SLE), lupus erythematosus,cutaneous (CLE) or lupus nephritis of the lupus of wanting treatment target.The lupus for the treatment of can be early stage, mid-term or terminal illness in when beginning treatment.In the embodiment of treatment lupus nephritis, described lupus nephritis can be any in the I-VI level.For example, the lupus that treat can be II level lupus nephritis, III level lupus nephritis, IV level lupus nephritis or V level lupus nephritis.In one embodiment, behind described combination begin treatment, compare with albuminuria and/or activeness urinary sediment level that object before the treatment beginning shows, described object demonstrates that albuminuria alleviates and/or the activeness urinary sediment alleviates.For example, albuminuria can reduce at least 25%, at least 50%, at least 75% or at least 90%, or albuminuria can be brought down below 1g/ days or be lower than 500mg/ days, and/or the activeness urinary sediment can reduce at least 25%, at least 50%, at least 75% or at least 90%, perhaps after the treatment beginning activeness urinary sediment only arranged.

In [0016] one embodiment, before described combination begin treatment, described object demonstrates albuminuria, has improved this albuminuria by treatment.For example, before the treatment beginning, the albuminuria that described object demonstrates is higher than 500mg/ days, is higher than 1000mg/ days, is higher than 2000mg/ days or is higher than 3500mg/ days.After the treatment beginning, albuminuria can reduce at least 25%, at least 50%, at least 75% or at least 90%, or albuminuria can be brought down below 1g/ days or be lower than 500mg/ days.Another example is, before the treatment beginning, the protein that described object demonstrates is higher than 0.5 with the ratio of kreatinin, be higher than 1 or be higher than 2; After the treatment beginning, the protein in the object urine can reduce at least 25% or at least 50% with the ratio of kreatinin, and perhaps this ratio can be brought down below 1 or be lower than 0.5.

[0017] on the one hand, this method comprises with described non-expendable CD4 antibody and the described second compounds for treating object and to continue this object of treatment to continue relief of symptoms with described non-expendable CD4 antibody or with described second chemical compound (not making up each other) then with mitigation symptoms.For example, in a class embodiment, behind described combination begin treatment, lupus improves; Stop then with the described object of this combined therapy, but give the non-expendable CD4 antibody that this object is treated effective dose.In another kind of illustrative embodiments, behind described combination begin treatment, lupus improves; Stop then with the described object of this combined therapy, but give second chemical compound or one or more other chemical compounds that this object is treated effective dose.

[0018] another kind of conventional embodiment also provides the method for treatment mammalian object such as people's lupus nephritis.In the method, the non-expendable CD4 antibody with the treatment effective dose gives described object.Behind described non-expendable antibody begin treatment, to compare with albuminuria and/or activeness urinary sediment level that object before the treatment beginning shows, described object shows that renal function improves, albuminuria alleviates and/or the activeness urinary sediment alleviates.For example, albuminuria can reduce at least 25%, at least 50%, at least 75% or at least 90%, and perhaps albuminuria can be brought down below 1g/ days or be lower than 500mg/ days; Protein can reduce at least 25% or at least 50% with the ratio of kreatinin, and perhaps this ratio can be brought down below 1 or be lower than 0.5; And/or the activeness urinary sediment can reduce at least 25%, at least 50%, at least 75% or at least 90%, perhaps after the treatment beginning activeness urinary sediment only arranged.

[0019] described lupus nephritis can be any in the I-VI level.For example, the lupus that treat can be II level lupus nephritis, III level lupus nephritis, IV level lupus nephritis or V level lupus nephritis.

In [0020] one embodiment, before the treatment beginning, described object demonstrates albuminuria and is higher than 500mg/ days, is higher than 1000mg/ days, is higher than 2000mg/ days or is higher than 3500mg/ days.In one embodiment, for example reduced at least 25%, at least 50%, at least 75% or at least 90% with albuminuria behind the described antibody begin treatment, or be brought down below 1g/ days or be lower than 500mg/ days.In one embodiment, for example reduced at least 25% or at least 50%, or be brought down below 1 or be lower than 0.5 with the ratio of protein behind the described antibody begin treatment and kreatinin.

[0021] described non-expendable CD4 antibody can be selected from down group: the antibody that a) comprises heavy chain amino acid sequence shown in light-chain amino acid sequence shown in the SEQ ID NO:3 and the SEQ ID NO:6; B) comprise the antibody of heavy chain amino acid sequence shown in light-chain amino acid sequence shown in the SEQ IDNO:9 and the SEQ ID NO:12; C) comprise the antibody of heavy chain amino acid sequence shown in light-chain amino acid sequence shown in the SEQ ID NO:15 and the SEQ ID NO:18; D) comprise the antibody of heavy chain amino acid sequence shown in light-chain amino acid sequence shown in the SEQ ID NO:21 and the SEQ ID NO:24; E) comprise a), b), c) or the antibody of the CD4 binding fragment of antibody d); F) comprise the antibody of the CDR3 (SEQ ID NO:27) of light chain shown in Figure 1A; G) comprise the antibody of the CDR3 (SEQ ID NO:30) of heavy chain shown in Fig. 1 D; H) comprise CDR1, the CDR2 of light chain shown in Figure 1A and the antibody of CDR3 (SEQ ID NO:25-27); I) comprise CDR1, the CDR2 of heavy chain shown in Fig. 1 D and the antibody of CDR3 (SEQ ID NO:28-30); And j) comprises CDR1, the CDR2 of heavy chain shown in CDR1, the CDR2 of light chain shown in Figure 1A and CDR3 and Fig. 1 D and the antibody of CDR3 (SEQ ID NO:25-30).Similarly, described antibody can be with Fig. 1-4 in arbitrary shown in the bonded CD4 antibody of identical epi-position of antibody.

[0022] for relevant, the optional combination of for example non-expendable antibody and at least a second chemical compound, antibody type and/or or the like, all features basically of said method also are applicable to these embodiments.For example, in an embodiment of the invention, described non-expendable CD4 antibody is optional to be humanized antibody, has nonglycosylated Fc part, not in conjunction with the Fc receptor, comprise the Cytotoxic aminoacid replacement that changes the Clq combination and/or depend on complement, comprise and remedy the receptors bind epi-position, comprise the serum albumin binding peptide, and/or have three or more antigen binding site.In some embodiment, described antibody is can be in conjunction with the anti--CD4 variant antibody of FcRN receptor.

The conventional embodiment of [0023] one class provides the method for the multiple sclerosis of treatment mammalian object such as human subjects.In the method, non-expendable CD4 antibody and/or at least a second chemical compound with the treatment effective dose gives described object.For example, the second suitable chemical compound for example includes, but not limited to cytotoxic agent; Immunosuppressant (for example, cyclophosphamide); B cell surface marker antagonist; The antibody of B cell surface marker; CD20 antibody (for example, Rituximab (Rituximab)); CD5, CD28 or CD40 antibody or blocker; 17-hydroxy-11-dehydrocorticosterone (for example, prednisone), CTLA4-Ig, α 4-alpha 2 integrin antibodies such as natalizumab (natalizumab)

Mycophenolate, his spit of fland, LFA-1 or CD-11a antibody or blocker, il-1 2 antibody, interferon-(for example, interferon beta-1a as

Or

Or interferon beta-1b as

Acetic acid glatiramer (glatiramer acetate)

CD52 antibody such as alemtuzumab (alemtuzuman) Interleukin-2-receptor antibody such as daclizumab (daclizumab) (

The antibody of interleukin-2 receptor alpha subunit), or the like.

[0024] embodiment of correlation type provides the method for certain disease of treatment mammalian object (for example, human subjects).Described disease can be rheumatoid arthritis, asthma, psoriasis, transplant rejection, graft versus host disease, multiple sclerosis, Crohn disease, ulcerative colitis, xerodermosteosis or other autoimmune conditions or disease.In the method, non-expendable CD4 antibody and at least a second combination of compounds with the treatment effective dose gives this object.In a class embodiment, described second chemical compound is cyclophosphamide, mycophenolate or CTLA4-Ig.

[0025] for relevant, for example the type of antibody type, second chemical compound and/or or the like, all features basically of said method also are applicable to these embodiments.For example, described non-expendable CD4 antibody can be selected from down group: the antibody that a) comprises heavy chain amino acid sequence shown in light-chain amino acid sequence shown in the SEQ ID NO:3 and the SEQ ID NO:6; B) comprise the antibody of heavy chain amino acid sequence shown in light-chain amino acid sequence shown in the SEQ ID NO:9 and the SEQ ID NO:12; C) comprise the antibody of heavy chain amino acid sequence shown in light-chain amino acid sequence shown in the SEQ ID NO:15 and the SEQ ID NO:18; D) comprise the antibody of heavy chain amino acid sequence shown in light-chain amino acid sequence shown in the SEQ ID NO:21 and the SEQ ID NO:24; E) comprise a), b), c) or the antibody of the CD4 binding fragment of antibody d); F) comprise the antibody of the CDR3 (SEQ ID NO:27) of light chain shown in Figure 1A; G) comprise the antibody of the CDR3 (SEQ ID NO:30) of heavy chain shown in Fig. 1 D; H) comprise CDR1, the CDR2 of light chain shown in Figure 1A and the antibody of CDR3 (SEQ ID NO:25-27); I) comprise CDR1, the CDR2 of heavy chain shown in Fig. 1 D and the antibody of CDR3 (SEQ ID NO:28-30); And j) comprises CDR1, the CDR2 of heavy chain shown in CDR1, the CDR2 of light chain shown in Figure 1A and CDR3 and Fig. 1 D and the antibody of CDR3 (SEQ ID NO:25-30).Similarly, described antibody can be with Fig. 1-4 in arbitrary shown in the bonded CD4 antibody of identical epi-position of antibody.

The accompanying drawing summary

[0026] Figure 1A-1F has shown the heavy chain of an embodiment of the non-expendable CD4 antibody of TRX1 and the nucleotide and the aminoacid sequence of light chain.Figure 1A has shown the nucleotide (SEQ ID NO:1) and aminoacid (the SEQ ID NO:2) sequence of light chain, and CDR and framework region.Figure 1B has shown the nucleotide sequence (SEQ ID NO:1) of light chain.Fig. 1 C has shown to be had (SEQ ID NO:2) and do not have the aminoacid sequence of the light chain of (SEQ ID NO:3) targeting sequencing.Fig. 1 D has shown the nucleotide (SEQ ID NO:4) and aminoacid (the SEQ ID NO:5) sequence of heavy chain, and CDR and framework region.Fig. 1 E has shown the nucleotide sequence (SEQ ID NO:4) of heavy chain.Fig. 1 F has shown to be had (SEQ ID NO:5) and do not have the aminoacid sequence of the heavy chain of (SEQ ID NO:6) targeting sequencing.

[0027] Fig. 2 A-2F has shown the heavy chain of an embodiment of the non-expendable CD4 antibody of TRX1 and the nucleotide and the aminoacid sequence of light chain.Fig. 2 A has shown the nucleotide (SEQ ID NO:7) and aminoacid (the SEQ ID NO:8) sequence of light chain, and CDR and framework region.Fig. 2 B has shown the nucleotide sequence (SEQ ID NO:7) of light chain.Fig. 2 C has shown to be had (SEQ ID NO:8) and do not have the aminoacid sequence of the light chain of (SEQ ID NO:9) targeting sequencing.Fig. 2 D has shown the nucleotide (SEQ ID NO:10) and aminoacid (the SEQ ID NO:11) sequence of heavy chain, and CDR and framework region.Fig. 2 E has shown the nucleotide sequence (SEQ ID NO:10) of heavy chain.Fig. 2 F has shown to be had (SEQ ID NO:11) and do not have the aminoacid sequence of the heavy chain of (SEQ ID NO:12) targeting sequencing.

[0028] Fig. 3 A-3F has shown the heavy chain of an embodiment of the non-expendable CD4 antibody of TRX1 and the nucleotide and the aminoacid sequence of light chain.Fig. 3 A has shown the nucleotide (SEQ ID NO:13) and aminoacid (the SEQ ID NO:14) sequence of light chain, and CDR and framework region.Fig. 3 B has shown the nucleotide sequence (SEQ ID NO:13) of light chain.Fig. 3 C has shown to be had (SEQ ID NO:14) and do not have the aminoacid sequence of the light chain of (SEQ ID NO:15) targeting sequencing.Fig. 3 D has shown the nucleotide (SEQ ID NO:16) and aminoacid (the SEQ ID NO:17) sequence of heavy chain, and CDR and framework region.Fig. 3 E has shown the nucleotide sequence (SEQ ID NO:16) of heavy chain.Fig. 3 F has shown to be had (SEQ ID NO:17) and do not have the aminoacid sequence of the heavy chain of (SEQ ID NO:18) targeting sequencing.

[0029] Fig. 4 A-4F has shown the heavy chain of an embodiment of the non-expendable CD4 antibody of TRX1 and the nucleotide and the aminoacid sequence of light chain.Fig. 4 A has shown the nucleotide (SEQ ID NO:19) and aminoacid (the SEQ ID NO:20) sequence of light chain, and CDR and framework region.Fig. 4 B has shown the nucleotide sequence (SEQ ID NO:19) of light chain.Fig. 4 C has shown to be had (SEQ ID NO:20) and do not have the aminoacid sequence of the light chain of (SEQ ID NO:21) targeting sequencing.Fig. 4 D has shown the nucleotide (SEQ ID NO:22) and aminoacid (the SEQ ID NO:23) sequence of heavy chain, and CDR and framework region.Fig. 4 E has shown the nucleotide sequence (SEQ ID NO:22) of heavy chain.Fig. 4 F has shown to be had (SEQ ID NO:23) and do not have the aminoacid sequence of the heavy chain of (SEQ ID NO:24) targeting sequencing.

[0030] Fig. 5 be the NZBxW F1 of SLE clinical before in the efficacy models progression of disease with the sketch map of change of age.

[0031] Fig. 6 A-6F has illustrated to giving the reaction of non-expendable CD4 antibody.Fig. 6 A is the diagram of time to progress (albuminuria is 300mg/dl or death), Fig. 6 B is the function of survival percent with the treatment beginning back time, Fig. 6 C is the albuminuria of treatment 5 months the time, and Fig. 6 D is average blood urea nitrogen and the function of treatment beginning back time, begin treatment when animal 8 months is big.Fig. 6 E has shown the relation of time to progress (albuminuria is 300mg/dl), and Fig. 6 F has shown the function of survival percent and treatment beginning back time, begin treatment when animal 6 months is big.

[0032] Fig. 7 A-7B is for reversing the diagram of seriousness lupus nephritis with non-expendable CD4 Antybody therapy.Fig. 7 A has shown the percentage ratio of sign mice of albuminuria＜300mg/dl during the time after treatment.Fig. 7 B has shown at the percentage ratio for the treatment of the mice of reversing in first month from albuminuria 〉=300mg/dl.

[0033] Fig. 8 A-8D is the diagram to the reaction that gives non-expendable CD4 antibody.Fig. 8 A has shown the ds-DNA antibody titer when raising, and Fig. 8 B has shown the titre for the treatment of after 3 months.Fig. 8 C has shown the number for the treatment of the CD4+CD69+ cell of finding after 3 weeks in spleen.Fig. 8 D has shown the number for the treatment of the CD4+CD25+ cell of finding after 3 weeks in spleen.

[0034] Fig. 9 A-9B has shown treatment albuminuretic multiple comparisons analysis in the time of 6 months, Fig. 9 A employing cyclophosphamide ( ) the treatment group organizes in contrast, Fig. 9 B adopts the non-expendable Antybody therapy group of CD4 to organize in contrast.

[0035] sketch map of Figure 10 for recurring in time and disappear by the inductive EAE progression of disease of SJL/J injected in mice PLP peptide to efficacy models before clinical as MS.

[0036] Figure 11 A-11B is the diagram to the reaction that gives non-expendable CD4 antibody.Figure 11 A be control antibodies, acetic acid glatiramer ( ), the time dependent diagram of clinical score of α-4 alpha 2 integrin antibodies, CTLA4-Ig and non-expendable CD4 Antybody therapy group.Figure 11 B has shown average every day of the clinical score of these groups.

[0037] Figure 12 A-12B is the diagram to the reaction that gives non-expendable CD4 antibody.Figure 12 A is the time dependent diagram of clinical score of control antibodies, CTLA4-Ig and non-expendable CD4 Antybody therapy group.Figure 12 B has shown average every day of the clinical score of these groups.

[0038] Figure 13 A-13B is the diagram to the reaction that gives non-expendable CD4 antibody.Figure 13 A is the time dependent diagram of clinical score of control antibodies, CTLA4-Ig and non-expendable CD4 Antybody therapy group.Figure 13 B has shown average every day of the clinical score of these groups.

[0039] Figure 14 has showed the spinal cord slice of the mice of control antibodies or CD4 Antybody therapy, shows that non-expendable CD4 Antybody therapy has reduced the demyelination of EAE.

[0040] Figure 15 has shown with ICOS in the animal per microliters of blood of control antibodies, non-expendable CD4 antibody or CTLA4-Ig treatment ^HiCD4 or ICOS ^HiThe number of cd8 t cell.

[0041] Figure 16 has compared with the clinical score of non-expendable CD4 antibody, expendable (depleting) CD4 antibody, control antibodies, CTLA4-Ig or expendable CD8 Antybody therapy myelin oligodendrocyte glycoprotein (MOG)-inducing peptide EAE situation of change in time.

[0042] Figure 17 A-17B is the diagram to the reaction that gives non-expendable CD4 antibody.Figure 17 A has shown the percentage ratio that indicates the mice of moment albuminuria＜300mg/dl after treatment.Figure 17 B has shown the percentage ratio of the mice of reversing from albuminuria 〉=300mg/dl.

[0043] Figure 18 A-18D is the diagram to the reaction of treatment.Figure 18 A is the relation of time and progression of disease (albuminuria 300mg/dl or death), Figure 18 B is for survival percent of the animal of the combined therapy of non-expendable CD4 antibody and 50mg/kg/ days MMF and the function of treatment beginning back time, is shown in Figure 18 C and Figure 18 D respectively with time and the progression of disease of the animal of the combined therapy of non-expendable CD4 antibody and 25mg/kg/ days MMF and the relation of the percent of surviving.

[0044] Figure 19 A-19B has shown treatment albuminuretic multiple comparisons analysis in the time of 2 months, adopts control antibodies treatment group to organize in contrast.With the group of 50mg/kg MMF every day (separately inclusive NAND expendable CD4 antibody combination) treatment the results are shown in Figure 19 A, and the results are shown in Figure 19 B with group that every day, 25mg/kg MMF treated.

[0045] Figure 20 A-20I is the diagram to the reaction of treatment.Shown among the figure and used control antibodies, non-expendable CD4 antibody, indicate the MMF of dosage, or non-expendable CD4 antibody and indicate the number (Figure 20 A) of CD4+T cell in every microliters of blood of animal of combined therapy of MMF of dosage, the number of B2B cell in every microliters of blood (Figure 20 B), the number of CD4+T cell in each spleen (Figure 20 C), the number of B2B cell in each spleen (Figure 20 D), the plasmacytic number of IgM+ (Figure 20 E), isotype-change plasmacytic number (Figure 20 F), the number of germinal center's cell (Figure 20 G), the number (Figure 20 H) of each spleen mesoplasmatocyte sample dendritic cell, and the MHC II expression (Figure 20 I) of plasma cell sample dendritic cell.

[0046] Figure 21 A-21B has shown nucleic acid and the aminoacid sequence of people CD4.Figure 21 A has shown the people CD4 aminoacid sequence of the mature protein that targeting sequencing is cut.Figure 21 B has shown sophisticated people CD4DNA sequence.

Definition

[0047] except as otherwise noted, technology used herein and scientific terminology and the technical field of the invention Those skilled in the art's common understanding has identical implication. To give a definition the understanding of this area is mended Fill, relate to the application but be not attributed to any relevant or irrelevant situation, for example any total patent or application. With in the literary composition those any method similar or of equal value is described and material all can be used for practical test the present invention, literary composition In nonrestrictive materials and methods has been described. Therefore, the term that adopts in the literary composition is only in order to describe particular implementation Mode and nonrestrictive.

[0048] in this specification and additional claims, singulative " " comprises that plural number refers to, Except offering some clarification in addition in the literary composition. Therefore, for example, when mentioning " protein ", comprise a plurality of protein; Comprise a plurality of cells when mentioning " cell ", the rest may be inferred.

[0049] " lupus " in the text reference and autoimmune disease or disease of attacking the antibody of connective tissue Disease. The principal mode of lupus is systemic lupus (SLE), and this disease also can be involved skin. " lupus " in the text Comprise SLE and other types lupus (comprise, for example, lupus erythematosus,cutaneous (CLE), lupus nephritis (LN), the outer lupus of kidney, encephalitis lupus, paediatrics lupus, non-kidney lupus, discoid lupus and alopecia lupus).

[0050] " object " is often referred to the people in the text, but also can be inhuman mammal. Exemplary is inhuman Mammal comprises laboratory animal, domestic animal, pet, sports animal and domestic animal, as mouse, cat, Dog, Ma Heniu. Usually, can treat described object, for example treat autoimmune conditions, group Knit and transplant relevant treatment, etc. On the one hand, can carry out the lupus treatment to this object. For this paper Purpose, this qualified object just experiencing or once lived through one or more lupus symptom, symptom or Other signs or diagnosed out lupus (for example, can newly diagnose out, or diagnose out before New outbreak is again, or the again newly outbreak of chronic steroid-dependent, or the lupus of suffering from risk arranged). Meet The object of lupus treatment condition can be by kidney biopsy screening and/or by test example such as following those autoantibodies Experiment sieving identify that wherein, the generation of autoantibody is by qualitative, preferred qualitative assessment. Exemplary The SLE related auto-antibodies is anti--antinuclear antibodies (Ab), anti--double-stranded DNA (dsDNA) Ab, anti--Sm Ab, anti--nuclear ribonucleoprotein Ab, anti--phosphatide Ab, anti--ribosomes P Ab, anti--Ro/SS-A Ab, anti--Ro Ab With anti--La Ab.

[0051] diagnosis of lupus (and suitable lattice of determining treatment) can be carried out according to means known in the art. For example, can diagnose SLE according to the standard of U.S. rheumatology association (ACR). Can be by the The British Isles wolf " A " standard of sore active organization (BILAG, British Isles Lupus Activity Group) or two BILAG " B " standard is come the definition of activities disease, for example, and such as " the Method for that is entitled as of Brunetta Treating lupus (lupus methods for the treatment of) " the mark that adopts of U.S. Patent Application Publication No. 2006/0024295 Accurate. Tan etc., (1982) " The 1982 Revised Criteria for the Classification of SLE (1982 The SLE criteria for classification of year revision) " Arth Rheum 25:1271-1277 adopts is used for diagnosing SLE's Some symptom, symptom or other signs can be: the rash on malar rass such as the cheek, plate-like rash or projection Erythema, photosensitivity is such as the reaction (causing fash to produce or deterioration) to daylight, in canker sore such as nose or the mouth Usually bitterly ulcer not, arthritis as relate to two or more ends slightly the Non-erosive arthritis in joint (close The not destroyed arthritis of bone around the joint), scrositis, pleurisy or pericarditis, ephrosis is as having in the urine Too much protein (albuminuria, the demonstration of test cotton swab is higher than 0.5g (gram)/sky or 3+) and/or cell are tubular (cellular cast) (derived from unusual element of urine and/or leucocyte and/or renal tubular cell), the neurology disease Levy, symptom or other signs, epileptic attack (tic), and/or mental disease or known energy when not having medicine Cause the metabolic disorder of this impact and hematology symptom, symptom or other signs as hemolytic anemia or in vain Leukopenia (every cubic millimeter lencocyte count is lower than 4000 cells) or lymphocyte reduce (every cube The lymphocyte of millimeter is lower than 1500) or decrease of platelet (every cubic millimeter blood platelet is lower than 100000). Being bound to detect leucocyte minimizing and lymphocyte in two or more situations reduces. Do not exist known Induced drug the time be bound to detect decrease of platelet. The invention is not restricted to these symptom, the symptom of lupus Or other signs.

[0052] outbreak of ephritis lupus may be defined as: 1) Scr amplification in 1 month〉30%, or 2) ephrosis Syndrome recurrence or occur, or 3) urine protein increases by 3 times, i.e. the baseline albuminuria〉1g/24 hour or as U.S. Patent Application Publication No. 2006/0024295 is described. For lupus nephritis, can pass through according to United States Patent (USP) The ephritis of application publication number 2006/0024295 described kidney standard definition shows effect to confirm to treat suitable lattice.

[0053] lupus nephritis can be chosen wantonly and be diagnosed and be categorized as ISN/WHOI level, II level, III level, IV Level, V level or VI level lupus nephritis, for example, such as (2004) such as Weening at " The classification of Glomerulonephritis in systemic lupus erythematosus revisited is (to systemic loupus erythematosus Brightic classification during recurrence) " description among the Kidney International 65:521-530.

[0054] " treatment " of object refer in the text therapeutic treatment and preventative means. Need the right for the treatment of Resemble and comprise and suffer from lupus (or other symptoms or autoimmune conditions such as MS, rheumatoid arthritis, or scorching The property enteropathy) object and the object that needs prevention lupus (or other illnesss). Therefore, object can be by Diagnosis suffers from lupus (or other illnesss) or tends to or be easy to suffer from lupus (or other illnesss).

[0055] term " alleviation " refers to alleviate, reduce or eliminate certain situation, disease, illness or phenotype in the text, Comprise unusual or symptom.

[0056] " symptom " of disease or illness (for example, lupus) refers to that object experiences and indicates appointing of disease What ill phenomenon or structure, function or feel to depart from normal.

[0057] statement " treatment effective dose " refers to effective prevention, alleviation or treatment disease or illness (for example, wolf Sore, MS, rheumatoid arthritis, or inflammatory bowel disease) amount. For example, " the treatment effective dose " of antibody refers to Can effectively prevent, alleviate or treat the antibody amount of specified disease or illness. Similarly, antibody and second chemical combination " the treatment effective dose " of the combination of thing refers to can effectively prevent, alleviate or treat specified disease or illness when combination Antibody amount and the second compound amount.

[0058] " combination " that should be understood that two kinds of compounds do not refer to that these two kinds of compounds will be mixed with each other ground Give. Therefore, the mixture that utilizes or adopt this combined therapy to comprise to give each compound or give separately Each compound is included on the same day or not and gives on the same day. Therefore, term " combination " refers to separately or is each other mixed Use two or more compounds for treating with closing. For example when giving object with antibody and second compound combination The time, have in the subject that second compound also can be present in the object in the antibody, no matter described antibody and Two compounds are independent or mixing gives this object.

[0059] T4 antigen or " CD4 " are a kind of at T lymphocyte and some other cell surface expression Glycoprotein. CD4 other titles in the literature comprise differentiation a small bundle of straw, etc. for silkworms to spin cocoons on 4 and L3T4. CD4 is described in, example As, the online Meng in the upper human data storehouse (Man database) of WWW www.ncbi.nlm.nih.gov/Omim 186940 clauses and subclauses in the Dare heredity (Online Mendelian Inheritance).

[0060] " CD4 antibody " is the antibody with enough affinity and specific binding CD4. For example, Randomly, this antibody in conjunction with the affinity of CD4 and specificity and TRX1 antibody to CD4 in conjunction with affine Power and specificity are quite or substantially similar. In the literary composition, " CD4 antibody ", " anti-CD 4 antibodies " and " anti--CD4 " Equivalent terms and replaceable use.

[0061] " non-expendable CD4 antibody " consumes and is less than 50% CD4+ cell, preferably is lower than 25%CD4+ Cell most preferably is lower than the CD4 antibody of 10% CD4+ cell. On the contrary, " expendable CD4 antibody " is Consume 50% or more CD4+ cells, perhaps in addition 75% or more or this 90% or more CD4+ cells CD4 antibody. The consumption of CD4+ cell (for example, circulation CD4+ cellular level in the Antybody therapy object Reduce) can obtain by various mechanism, the cytotoxicity such as the antibody dependent cellular mediation depends on complement Cytotoxicity, the inhibition of T cell proliferation, and/or the inducing of T cell death.

[0062] term " antibody " uses with broad sense in the text, specifically comprises monoclonal antibody, many grams Grand antibody, the multi-specificity antibody (for example bispecific antibody), the inosculating antibody that are formed by at least two complete antibodies Body, people's antibody and the antibody fragment that demonstrates required biologically active (for example, CD4 combination). Antibody is Comprise basically or a kind of or many by immunoglobulin gene or immunoglobulin gene fragment coding of part The protein of peptide species. Known immunoglobulin gene comprises κ, λ, α, γ, δ, ε and μ constant region Gene, and countless immune globulin variable region gene.

[0063] " antibody fragment " comprises the part of complete antibody, preferably comprises its antigen binding domain.The example of antibody fragment comprises Fab, Fab ', F (ab ') ₂With the Fv fragment; Bispecific antibody; Linear antibody; The single-chain antibody molecule; With the multi-specificity antibody that forms by antibody fragment.

[0064] " complete antibody " is to comprise heavy chain-and the antibody in light chain-variable domains and Fc district.

[0065] " natural antibody " normally gathers (heterotetrameric) glycoproteins by about 150,000 daltonian different four of two identical light (L) chain identical with two heavy (H) chains formations.Each light chain links to each other with heavy chain by a covalent disulfide bonds, and the disulfide bond number difference between the different immunoglobulin isotype heavy chain.Each heavy chain and light chain also have disulphide bridges in the regularly spaced chain.One end of each heavy chain is variable domains (V _H), follow a plurality of constant domain.One end of each light chain is variable domains (V _L), the other end is a constant domain; The constant domain of light chain is alignd with first constant domain of heavy chain, and the light chain variable domain aligns with the variable domains of heavy chain simultaneously.It is believed that particular amino acid residue has formed the interface between light chain and the weight chain variable domain.

[0066] term " variable " refers to that the sequence of some part of variable domains alters a great deal and can be used for combining and this fact of specificity of each specific antibodies and its specific antigen between antibody.Yet transmutability is not equally distributed in the variable domains of antibody.It concentrates on three sections that are called hypervariable region in light chain and the weight chain variable domain.The more conservative part of variable domains is called framework region (FR).The variable domains of natural heavy chain and light chain respectively comprises by continuous four FR (taking β-lamella configuration) of three hypervariable regions (formation is connected the ring of β-lamellar structure, and forms part β-lamellar structure in some cases) more.Hypervariable region in each chain is closely linked by FR, and combine with the hypervariable region of other chains, the antigen binding site that forms antibody is (referring to Kabat etc., Sequences of Proteins of Immunological Interest (sequence of interested immune protein), the 5th edition, Public Health Service, National Institutes ofHealth (public health service department of NIH), Bethesda (Bei Sida), Md. (Maryland State) (1991)).Constant domain is not participated in antibody directly and is combined with antigenic, but demonstrates various effector functions, participates in the cytotoxicity of antibody dependent cellular mediation as antibody.

[0067] papain digestion antibody produces two identical antigen-binding fragments, is called " Fab " fragment, and they have an antigen binding site and remaining " Fc " fragment separately, and this title reflects that it is easy to crystallization.Pepsin produces F (ab ') ₂Fragment, this fragment have two antigen binding sites and can with antigen crosslinking.

[0068] " Fv " is the minimum antibody fragment that contains complete antigen recognition and antigen binding site.This zone is made of tight, the heavy chain of non-covalent connection and the dimer of a light chain variable domain.In this configuration, thereby three hypervariable regions of each variable domains interact at V _H-V _LThe dimer surface forms antigen binding site.In a word, six hypervariable regions antigen-binding specificity of having given antibody.Yet, even if variable domains half Fv of the hypervariable region of three antigenic specificities (or only comprise) also can discern and conjugated antigen, although its affinity is lower than complete binding site.

[0069] the Fab fragment also contains the constant domain of light chain and first constant domain (CH1) of heavy chain.The segmental difference of Fab ' fragment and Fab is to have added some residues at the carboxyl terminal of heavy chain CH1 domain, comprising one or more cysteine of antibody hinge region.Fab '-SH is called as Fab ' in the text, and wherein the cysteine residues of constant domain has at least one free sulfhydryl groups.F (ab ') ₂Antibody fragment is formed by a pair of Fab ' fragment at first, has the hinge cysteine between them.Other chemical couplings of antibody fragment also are known.The detailed description of relevant other antibody fragments can referring to, for example, Fundamental Immunology (basic immunology), W.E.Paul compile, Raven Press (Lei Fen publishing house), N.Y. (1999).

[0070] although various antibody fragment defines by the digestion complete antibody, the technical staff will understand, and also can or utilize these fragments of recombinant DNA method de novo synthesis by chemical method.Therefore, term " antibody " comprises antibody or its fragment by modifying complete antibody or utilizing recombinant DNA method de novo synthesis to produce in the text.

[0071] " light chain " of any vertebrates antibody (immunoglobulin) can be appointed as the two kinds of types that can clearly distinguish according to the aminoacid sequence of their constant domain, is called κ and λ, in a kind of.

[0072] aminoacid sequence according to the heavy chain constant domain can be divided into variety classes with antibody.Complete antibody mainly contains five classes: IgA, IgD, IgE, IgG and IgM, and some of them can further be divided into subclass (isotype), for example IgG1, IgG2, IgG3, IgG4, IgA and IgA2.Heavy chain constant domain corresponding to the different antibodies kind is called α, δ, ε, γ and μ.The subunit structure and the 3-d modelling of variety classes immunoglobulin are known.

[0073] " strand Fv " or " scFv " antibody fragment comprise the V of antibody _HAnd V _LDomain, wherein, these domains are present in the single polypeptide chain.Preferably, the Fv polypeptide is at V _HAnd V _LAlso comprise peptide linker between the domain, this makes scFv can form the required structure of conjugated antigen.The summary of scFv can be referring to the The Pharmacology of Monoclonal Antibodies (monoclonal antibody pharmacology) of Rosenburg and Moore volume, the 113rd volume, Springer-Verlag, New York (1994), the argumentation of Pluckthun in the 269-315 page or leaf.

[0074] term " bispecific antibody " refers to have the little antibody fragment of two antigen binding sites, and this fragment comprises in same polypeptide chain and light chain variable domain (V _L) continuous weight chain variable domain (V _H) (V _H-V _L).Be short to by use and can not form paired joint between two domains of same chain, this domain is forced to the complementary structure territory pairing of another chain and forms two antigen binding sites.Bispecific antibody is more complete to be described in, and for example, EP 404097; WO 1993/11161; With Hollinger etc., Proc.Natl.Acad.Sci.USA, 90:6444-6448 (1993).

[0075] term " monoclonal antibody " refers in the text from the antibody of the antibody population acquisition of basic homogenizing, it is identical and/or in conjunction with identical epi-position promptly to constitute each antibody of this colony, but may produce variant when the preparation monoclonal antibody, this variant exists with minimum usually.With polyclonal antibody goods (generally including the different antibodies at different determiners (epi-position)) difference, each monoclonal antibody is only at a determiner on the antigen.Except their specificity, the advantage of monoclonal antibody is that they pollute other immunoglobulins.Modifier " monoclonal " shows that the feature of this antibody is to obtain from the antibody population of basic homogenizing, but can not be interpreted as and need prepare this antibody by any ad hoc approach.For example, the monoclonal antibody that the present invention uses can be by the earliest by Kohler etc., Nature, and the hybridoma manufactured that 256:495 (1975) describes perhaps can be passed through recombinant DNA manufactured (referring to, for example, U.S. Patent number 4816567)." monoclonal antibody " for example also can adopt Clackson etc., Nature, and 352:624-628 (1991) and Marks etc., J.Mol.Biol., the technology that 222:581-597 (1991) describes is separated from phage antibody library.

[0076] monoclonal antibody described here specifically comprises " chimeric " antibody (immunoglobulin), wherein a part of heavy chain and/or light chain with derived from particular types or belong to the identical or homology of corresponding sequence in the antibody of specific antibodies classification or subclass, but the remainder of chain with derived from another kind or belong to the identical or homology of corresponding sequence in the antibody of another antibody classification or subclass, and the fragment that comprises this antibody, as long as they demonstrate required biological activity (U.S. Patent number 4816567; Morrison etc., Proc.Natl.Acad.Sci.USA, 81:6851-6855 (1984)).In the literary composition, interested chimeric antibody comprises " primatesization (primatized) " antibody, it comprises variable domains antigen binding sequence and human constant region sequence (U.S. Patent number 5693780) derived from non-human primates (for example old world monkey, as baboon, Rhesus Macacus or macaque).

[0077] " humanization " form of inhuman (for example, Muridae) antibody belongs to chimeric antibody, and it contains few sequence derived from non-human immunoglobulin.The major part of humanized antibody is human normal immunoglobulin's (receptor's antibody), and wherein the residue of receptor's hypervariable region is substituted as mice, rat, rabbit or residue with non-human primates hypervariable region of required specificity, affinity and ability by inhuman kind (donor antibody).In the certain situation, human normal immunoglobulin's framework region (FR) residue is substituted by corresponding inhuman residue.In addition, humanized antibody can comprise the residue of not finding in receptor's antibody or donor antibody.Make these and modify further optimization antibody performance.Usually, humanized antibody will comprise basically all or at least one, be generally two variable domains, wherein all or all basically hypermutation ring are corresponding to the hypermutation ring of non-human immunoglobulin, all or all basically FR are the FR of human normal immunoglobulin's sequence, but except the above-mentioned FR replacement.Also optional at least a portion constant region for immunoglobulin, normally the human normal immunoglobulin's constant region of comprising of humanized antibody.The visible Jones of more detailed description etc., Nature 321:522-525 (1986); Riechmann etc., Nature 332:323-329 (1988); And Presta, Curr.Op.Struct.Biol.2:593-596 (1992).

[0078] term " hypervariable region " is meant the amino acid residue of being responsible for the bonded antibody of antigen when being used for this paper.Hypervariable region comprise from the amino acid residue of " complementary determining region " or " CDR " (referring to, for example, Kabat etc., Sequences of Proteins of Immunological Interest (sequence of interested immune protein), the 5th edition, Public Health Service, National Institutes of Health (public health service department of NIH), Bethesda (Bei Sida), Md. (Maryland State) (1991)) and/or from those residues of " hypermutation ring " (referring to, for example, Chothia and Lesk J.Mol.Biol.196:901-917 (1987))." framework " or " FR " residue is those variable domains residues the defined hypervariable region residue in literary composition.

[0079] term " Fc receptor " and " FcR " are used for describing the receptor in binding antibody Fc district.The summary of FcR is seen Ravetch and Kinet (1991) Annu.Rev.Immunol 9:457-92; Capel etc., (1994) Immunomethods 4:25-34; With de Haas etc., (1995) J.Lab.Clin.Med.126:330-41.Other FcR comprise following those that identify, are also included within the middle of the term used herein " FcR ".This term also comprises the neonate receptor, FcRn, and it is responsible for the IgG of parent is transferred to fetus (Guyer etc., (1976) J.Immunol.117:587 and Kim etc., (1994) J.Immunol.24:249).

[0080] " the CD4 binding fragment " of antibody is the antibody fragment that keeps in conjunction with the ability of CD4.As mentioned above, this fragment is optional by digesting the generation of complete antibody or de novo synthesis.

[0081] " epi-position " is the specific region of binding antibody on the antigen molecule.

[0082] phrase " similar substantially " or " basic identical " refer in the text two numerical value (common one relevant with antibody of the present invention, another is relevant with reference/comparison antibody) between similarity enough high, thereby just (for example by described value, the Kd value) biological property that records, those skilled in the art think that the difference of these two values on biology and/or statistical significance is minimum or do not have.As the function of the value of reference/comparison antibody, it is about 50% that the difference between described two values preferably is lower than, and preferably is lower than approximately 40%, preferably is lower than approximately 30%, preferably is lower than approximately 20%, preferably is lower than about 10%.

[0083] " binding affinity " is often referred to the intensity of noncovalent interaction summation between the single binding site of molecule (for example, antibody) and its binding partners (for example, antigen).Except as otherwise noted, in the literary composition, " binding affinity " refers to inherent binding affinity, and it has reacted in conjunction with the 1:1 between (for example, antibody and the antigen) member is interacted.Molecule X uses dissociation constant (Kd) expression usually to the affinity of its companion Y.Affinity can be measured by conventional method known in the art, comprising those methods as herein described.The common slow fixation antigen of low-affinity antibody also is easy to dissociated trend, and the usually very fast conjugated antigen of high-affinity antibody and the long-term bonded trend of maintenance is arranged.The whole bag of tricks of measuring binding affinity is known in the art, and wherein any all can be used for purpose of the present invention.Concrete illustrative embodiments is described hereinafter.

In [0084] one embodiment, " Kd " of the present invention or " Kd value " adopts the Fab form of antibody interested and antigen measuring thereof by radiolabeled antigen in conjunction with test (RIA), this test is described by following test and is carried out: the antigen balance Fab that uses [the 125I]-labelling of Cmin when having the titration series of unlabelled antigen, catch bonded antigen and detect Fab with the anti--flat board of Fab antibody-Bao quilt then antigenic resolution binding affinity (solution binding affinity) (Chen etc., (1999) J.Mol Biol 293:865-881).For setting up experimental condition, catch anti--Fab antibody (Cappel Labs) bag with the 5ug/ml of 50mM sodium carbonate (pH9.6) preparation and spent the night by titer plate (Dynex), subsequently with 2% (w/v) bovine serum albumin of PBS preparation room temperature (about 23 ℃) sealing 2-5 hour.Go up in non-absorption dull and stereotyped (Nunc #269620) and to mix 100pM or 26pM[125I]-serial dilution of antigen and Fab interested is (for example, with Presta etc., the assessment unanimity of anti-VEGF antibodies, Fab-12 among (1997) Cancer Res.57:4593-4599).Then interested Fab is cultivated and spend the night; Yet can continue to cultivate the long period (for example, 65 hours) to guarantee to reach balance.Afterwards, mixture is transferred to caught dull and stereotyped room temperature and cultivate (for example, cultivating 1 hour).Remove solution then and with 0.1% Tween-20 of PBS preparation with flat board washing 8 times.Flat board is added 150ul/ hole scintillator (MicroScint after drying ^TM-20; Packard), use

Dull and stereotyped 10 minutes of γ numeration instrument (Packard) counting.20% each the being at war with property of Fab concentration that select to obtain to be less than or equal to maximum combined is in conjunction with test.According to another embodiment, Kd or Kd value are to adopt surperficial plasmon resonance test to measure, and are determined at 25 ℃ and carry out, and use

-2000 or

-3000 (BIAcore, Inc., Piscataway (Piscataway), New York) and immobilized antigen CM5 chips, reacton (RU) is about 10.In brief, the explanation according to supplier activates carboxymethylated glucosan biologic sensor chip (CM5, BIAcore Inc.) with N-ethyl-N '-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxy-succinamide (NHS).Antigen 10mM sodium acetate, pH4.8 are diluted to 5ug/ml (about 0.2uM), then the reacton (RU) of flow velocity injection to obtain about 10 coupling proteins that divides with 5ul/.Inject the 1M ethanolamine after the injections of antigens with the sealing unreacted radical.During kinetic measurement, the flow velocity that divides with about 25ul/ in the time of 25 ℃ is injected into the twice serial dilution (0.78nM-500nM) of Fab among the PBS that contains 0.05% Tween 20 (PBST).With Langmuir combination model simply one to one (

Evaluation software, version 3 .2) come calculations incorporated speed (kon) and the speed of dissociating (koff) by the while match combination or the sensorgram that dissociates.Equilibrium dissociation constant (Kd) is calculated as koff/kon ratio.Referring to, for example, Chen etc., (1999) J.Mol Biol 293:865-881.If the association rate that calculates by above-mentioned surperficial plasmon resonance test surpasses 10 ⁶M ^-1S ^-1Then can adopt fluorescent quenching technical measurement association rate, this technology is utilized 25 ℃ of photometer measurements, when having the antigen that concentration edges up the 20nM of PBS (pH7.2) preparation anti--fluorescent emission intensity of antigen-antibody (Fab form) increases or reduces and (excite=295nm; Emission=340nm, the 16nm band is logical), described spectrometer is as spectrophotometer (AvivInstruments) that cut-off equipment is housed or 8000 series with stirring cuvette

Spectrophotometer (Thermo Spectronic).

[0085] " aminoacid sequence " is the polymer (protein, polypeptide etc.) of amino acid residue or the character string of represented amino acid polymer, based on context decides.

[0086] term " immunosuppressant " is meant when referring to treat in the text and is used for suppressing or sheltering the mammiferous immune material of being treated.It comprises that suppressing cytokine produces, reduces or suppress autoantigen and express or shelter the antigenic material of MHC.The example of this reagent comprises the pyrimidine (referring to U.S. Patent number 4665077) that 2-amino-6-aryl-5-replaces; Nonsteroidal anti-inflammatory agent (NSAID); Ganciclovir, tacrolimus, glucocorticoids such as cortisone or aldosterone, antiinflammatory such as cyclooxygenase-2 inhibitor, 5-fat oxidation enzyme inhibitor, or LTRA; Purine antagonist such as azathioprine or mycophenolate (MMF); Alkylating agent such as cyclophosphamide; Bromocriptine; Danazol; Dapsone; Glutaraldehyde (sheltering MHC antigen, as described in U.S. Patent number 4120649); MHC antigen and MHC are segmental to be resisted-idiotype antibody; Cyclosporin A; Steroid such as 17-hydroxy-11-dehydrocorticosterone or glucocorticoid or glucocorticoid analogue, for example, prednisone, methylprednisolone and dexamethasone; Dihydrofolate reductase inhibitor such as methotrexate (oral or subcutaneous); Hydroxyl clorotepine (hydroxycloroquine); Sulfasalazine; Leflunomide; Cytokine or cytokine receptor antibody comprise anti--interferon-' alpha ',-β, or-gamma antibodies, Anti-tumor necrosis factor-alpha antibody (infliximab or adalimumab), anti-TNF-alpha immunization adhesin (Embrel), Anti-tumor necrosin-β antibody, anti--interleukin-2 antibody and anti--IL-2 receptor antibody; Anti--LFA-1 antibody, comprise anti--CD11a and anti--CD18 antibody; Allos resists-the lymphocyte globulin; General-T antibody, preferred resisting-CD3; The soluble peptide (1990/7/26 WO 1990/08187 that announces) that contains the LFA-3 binding structural domain; Streptokinase; TGF-β; Streptodornase; Host's RNA or DNA; FK 506; RS-61443; Deoxyspergualin; Rapamycin; TXi Baoshouti (Cohen etc., U.S. Patent number 5114721); T cell-receptor fragments (Offner etc., Science, 251:430-432 (1991); WO 1990/11294; Ianeway, Nature, 341:482 (1989); With WO 1991/01133); With T cell-receptor antibody (EP 340109) as T10B9.

[0087] term " cytotoxic agent " refers in the text to suppress or prevents cell function and/or cause the material of cytoclasis.This term comprises radiosiotope (At for example ²¹¹, I ¹³¹, I ¹²⁵, Y ⁹⁰, Re ¹⁸⁶, Re ¹⁸⁸, Sm ¹⁵³, Bi ²¹², P ³²Radiosiotope with Lu), chemotherapeutics, and the toxin of antibacterial, fungus, plant or animal origin such as micromolecule toxin or enzyme activity toxin, or its fragment.

[0088] " chemotherapeutics " is the chemical compound that is generally used for treating cancer.The example of chemotherapeutics comprises: alkylating agent as the plug for the group and

Cyclophosphamide; Alkylsulfonate such as busulfan, an improsulfan, and piposulfan; Aziridine such as benzo DOPA (benzodopa), carboquone, meturedepa (meturedopa), and uredepa (uredopa); Aziridine and methylene melamine (methylamelamine) comprise hexamethyl melamine, tretamine, triethylenephosphoramide (trietylenephosphoramide), triethylene thiophosphoramide (triethiylenethiophosphoramide) and trihydroxy first melamine (trimethylolomelamine); Acetogenin (especially bullatacin (bullatacin) and bullatacin ketone (bullatacinone)); Camptothecine (comprising the synthetic analogues hycamtin); Bryostatin; His spit of fland (callystatin), Cali; CC-1065 (comprising its adozelesin, carzelesin and bizelesin synthetic analogues); Latent algin (cryptophycins) (especially latent algin 1 and latent algin 8); Dolastatin; Many Ka-7038s (duocarmycin) (comprising synthetic analogues, KW-2189 and CB1-TM1); Soft coral alcohol; Water ghost any of several broadleaf plants alkali (pancratistatin); Sa Kediting (sarcodictyin); Sponge inhibin (spongistatin); Chlormethine such as chlorambucil, chlornaphazine, bile phosphamide (cholophosphamide), estramustine, ifosfamide, chlormethine, hydrochloric acid oxidation chlormethine, melphalan, novoembichin, phenesterin, prednimustine, trofosfamide, uracil mustard; Nitroso ureas (nitrosureas) is as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and Ranimustine; Antibiotic such as enediyne class antibiotic (enediyne antibiotics) (for example, calicheamycin, especially calicheamycin γ 1I and calicheamycin ω I1 (referring to, for example, Agnew, Chem Intl.Ed.Engl., 33:183-186 (1994)); Anthracycline antibiotics comprises anthracycline antibiotics A; Diphosphate is as clodronate; Ai Sipeila mycin (esperamicin); And neocarzinostain NCS chromophore and related color albumen enediyne class antibiotic chromophore, aklavine (aclacinomysin), D actinomycin D, Ao Simi star (authramycin), azaserine, bleomycin, actinomycin C, carubicin (carabicin), carminomycin, cardinophyllin, chromomycin (chromomycinis), actinomycin D, daunorubicin, detorubicin, 6-diazonium-5-oxygen-L-nor-leucine

Amycin (comprising morpholino-amycin, cyano group morpholino-amycin, 2-pyrrolin generation (pyrrolino)-amycin and deoxidation amycin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin such as ametycin, mycophenolic acid, nogalamycin, Olivomycin, peplomycin, porfiromycin (potfiromycin), puromycin, triferricdoxorubicin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; Anti--metabolism medicine such as methotrexate and 5-fluorouracil (5-FU); Folacin such as 9,10-dimethylpteroylglutamic acid, methotrexate, Pteropterin, trimetrexate; Purine analogue such as fludarabine, 6-mercaptopurine, ITG, thioguanine; Pyrimidine analogue such as ancitabine, azacytidine, 6-azauridine, carmofur, cytosine arabinoside, two BrdUs (dideoxyuridine), doxifluridine, enocitabine, floxuridine; Androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; Anti--epinephrine and aminoglutethimide, mitotane, trilostane; Folic acid supplement such as folinic acid (frolinic acid); Aceglatone; Aldophosphamide glucosides (aldophosphamide glycoside); Amino-laevulic acid; Eniluracil; Amsacrine; Doubly bent than new (bestrabucil); Bisantrene; Edatrexate; Defosfamide; Demecolcine; Diaziquone; Yi Fumi star (elformithine); Elliptinium acetate (elliptinium acetate); Epothilones (epothilone); Etoglucid; Ganite (Fujisawa).; Hydroxyurea; Mill is eaten polysaccharide; Lonidamine; Maytansinol class such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Mopidamol (mopidanmol); Nitragin; Pentostatin; Phenamet; Pirarubicin; Losoxantrone; Podophyllinic acid; 2-ethyl hydrazine; Procarbazine; Polysaccharides compound (JHS natural product company (JHS Natural Products), Eugene city, Oregon); Razoxane; Rhizomycin; Sizofiran; Spirogermanium; Tenuazonic acid; Triaziquone; 2,2 ', 2 ＂-trichloro-triethylamine; Trichothecenes (T-2 toxin especially, muconomycin (verracurin) A, Roridine A and peace general fourth (anguidine)); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Lid holder star (gacytosine); Cytosine arabinoside (" Ara-C "); Cyclophosphamide; Plug is for group; Taxane, for example,

Paclitaxel (executing expensive precious oncology company (Bristol-Myers Squibb Oncology), Princeton, New York during Bristol-U.S.), ABRAXAN ^TMDo not contain cremophor, the albumin through engineering approaches nanoparticle preparation of paclitaxel (united states drug companion company (AmericanPharmaceutical Partners), Shan Mubao (Schaumberg), Illinois) and

Docetaxel (RPR company (Rhone-Poulenc Rorer), Anthony (Antony), France); Chlorambucil (chloranbucil);

Gemcitabine; 6-thioguanine; Mercaptopurine; Methotrexate; Platinum analogs such as cisplatin and carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine;

Vinorelbine; Novantrone; Teniposide; Edatrexate; Daunorubicin; Aminopterin; Xeloda; Ibandronate; CPT-11; Topoisomerase enzyme inhibitor RFS 2000; Er Fujiajiniaoansuan (difluorometlhylornithine) (DMFO); Retinoid such as retinoic acid; Capecitabine; With above-mentioned any pharmaceutically acceptable salt, acid or derivant.

[0089] this definition also comprises anti--hormone preparation of adjusting or inhibitory hormone effect, as anti-estrogen class and selective estrogen receptor modulators (SERM), comprises that for example, tamoxifen (comprises Tamoxifen), raloxifene, droloxifene, the 4-hydroxy-tamoxifen, trioxifene, raloxifene (keoxifene), LYI17018, onapristone and Toremifene; Suppress the aromatase inhibitor of aromatase, it can regulate estrogenic generation among the adrenal gland, and 4 (5)-imidazoles are for example arranged, aminoglutethimide, Megestrol acetate,

Exemestane, Formestane (formestanie), fadrozole,

Vorozole, Letrozole and

Anastrozole; With anti--androgen such as flutamide, nilutamide, bicalutamide, leuprorelin, and goserelin; And troxacitabine (1,3-dioxolane nucleoside cytosine analog); Antisense oligonucleotide, especially those inhibition relate to the antisense oligonucleotide of gene expression in the signal transduction path of abnormal cell proliferation, as PKC-α, and Raf, and H-Ras; Vaccine such as gene therapy vaccine, for example,

Vaccine,

Vaccine and

Vaccine;

RIL-2;

Topoisomerase 1 inhibitor;

RmRH; With above-mentioned any pharmaceutically acceptable salt, acid or derivant.

[0090] term " cytokine " " be to act on the protein of other cells as the intercellular medium by what a kind of cell mass discharged.The example of this cytokine has: lymphokine, monokine; Interleukin (IL) is as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; Tumor necrosis factor such as TNF-α or TNF-β; Comprise LIF and kit part (KL) with other polypeptide factors.In the literary composition, the term cytokine comprises from natural origin or from the protein of reconstitution cell culture and the biological activity equivalent of native sequences cytokine, comprises synthetic micromolecule entity and pharmaceutically acceptable derivates and the salt that makes.

[0091] term " hormone " refers to usually by the excretory polypeptide hormone of the glandular organ with conduit.Hormone comprises, for example, and growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; Parathyroid hormone; Thyroxine; Insulin; Proinsulin; Relaxin; Relaxation precipitinogen (prorelaxin); Glycoprotein hormones such as follicle stimulating hormone (FSH), thyrotropin (TSH), and lutropin (LH); Prolactin antagonist, human placental lactogen, mice chorionic gonadotrophin related peptides, inhibin; Activin; Miller pipe mortifier; And thrombopoietin.In the text, the term hormone comprises from natural origin or from the protein of reconstitution cell culture and the biological activity equivalent of native sequences hormone, comprises synthetic micromolecule entity and pharmaceutically acceptable derivates and the salt that makes.

[0092] term " somatomedin " refers to promote the protein of growing, and it comprises, for example, and liver growth factor; Fibroblast growth factor; VEGF; Nerve growth factor such as NGF-β; Platelet derived growth factor; Transforming growth factor (TGF) is as TGF-α and TGF-β; Insulin like growth factor-1 and-II; Erythropoietin (EPO); Osteoinductive factors; Interferon such as interferon-' alpha ' ,-β and-γ; And colony stimulating factor (CSF) is as macrophage-CSF (M-CSF); Granular leukocyte macrophage-CSF (GM-CSF); And granulocyte-CSF (G-CSF).In the literary composition, the term somatomedin comprises from natural origin or from the protein of reconstitution cell culture and the biological activity equivalent of native sequences somatomedin, comprises synthetic micromolecule entity and pharmaceutically acceptable derivates and the salt that makes.

[0093] for the purpose of this paper, " tumor necrosis factor-alpha (TNF-α) " refers to humanTNF-'s molecule, and it comprises Pennica etc., Nature, 312:721 (1984) or Aggarwal etc., JBC, the described aminoacid sequence of 260:2345 (1985).

[0094] " TNF-alpha inhibitor " thus refer in the text generally by in conjunction with the TNF-α and its active reagent that suppresses the biological function of TNF-α to a certain extent that neutralizes.The example of tnf inhibitor specifically comprise in the text Embrel (

), infliximab ( ) and adalimumab (HUMIRA ^TM).

[0095] example of " nonsteroidal anti-inflammatory agent " or " NSAID " has aspirin, ibuprofen, and naproxen, indomethacin, sulindac, Tolmetin comprises its salt or derivant etc.

[0096] term " integrin " refer to allow cell combination and response extracellular matrix and participate in wound healing, cell differentiation, tumor cell is gone back to the nest and the receptor protein of various cell functions such as apoptosis.They are the parts that participate in the cell adhesion receptor extended familys of cell-extracellular matrix and cell-cell interaction.Functional integrin is made of two transmembrane glycoprotein subunits of non-covalent bonded α of being called and β.The α subunit all has certain homology each other, and the β subunit also is like this.Receptor always contains a α chain and a β chain.Example comprises α 6 β 1, α 3 β 1, α 7 β 1, LFA-1 etc.In the text, the term integrin comprises from natural origin or from the protein of reconstitution cell culture and the biological activity equivalent of native sequences integrin, comprises synthetic micromolecule entity and pharmaceutically acceptable derivates and the salt that makes." α 4-integrin " is at the α 4-β 1 of the expression of the leukocyte surface except that neutral leukocyte and α 4 subunits of α 4-β 7 integrins.

[0097] example of " integrin antagonist or antibody " comprises LFA-1 antibody in the text, as available from the sharp in accordance with the law pearl monoclonal antibody (efalizumab) of Genentech, Inc. (US) 460 Point San Bruno Blvd, South San Francisco, CA, 94080 (Genentech) ( ), or alpha-4 integrin antibody (for example, " α 4-alpha 2 integrin antibodies " is the antibody in conjunction with α 4-integrin) is as the natalizumab available from Baeyer King Company (Biogen)

Or diazacyclo phenylalanine derivative (WO2003/89410), (WO 2003/70709 for phenylalanine derivative, WO 2002/28830, WO2002/16329 and WO 2003/53926), benzyl propionate derivant (WO 2003/10135), enamine derivates (WO 2001/79173), propanoic derivatives (WO 2000/37444), alkane acid derivative (WO2000/32575), the phenyl derivatives (U.S. Patent number 6677339 and 6348463) that replaces, aromatic amine derivant (U.S. Patent number 6369229), ADAM disintegrin domain polypeptide (US 2002/0042368), α v β 3 alpha 2 integrin antibodies (EP 633945), azepine-bridge bicyclic amino acid derivative (WO 2002/02556), or the like.

[0098] " 17-hydroxy-11-dehydrocorticosterone " refers to that some have the general chemical constitution of steroid class, can simulate or strengthen any in the material of synthetic or natural generation of effect of 17-hydroxy-11-dehydrocorticosterone of natural generation.The example of synthetic 17-hydroxy-11-dehydrocorticosterone comprises prednisone, prednisolone (comprising methylprednisolone), dexamethasone triamcinolone, and betamethasone.

[0099] " B cell surface marker " or " B cell surface antigen " refer in the text the B cell surface expression, can be by the antigen of antagonist targeting bonded with it.Exemplary B cell surface marker comprises: CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85, (relevant description can be referring to The Leukocyte Antigen Facts Book (the true book series of human leucocyte antigen), second edition, 1997 with CD86 leukocyte surface labelling, volumes such as Barclay, academic press (Academic Press), (the Harcourt Brace ﹠amp of HB company; Co.), New York).Other B cell surface markers comprise: RP105, FcRH2, B cell CR2, CCR6, P2X5, HLA-DOB, CXCR5, FCER2, BR3, Btig, NAG14, SLGC16270, FcRH1, IRTA2, ATWD578, FcRH3, IRTA1, FcRH6, BCMA and 239287.Interested especially B cell surface marker is to compare mammiferous other non-B cell tissues preferentially at the labelling of expressing on the B cell, also can express on B cell precursor and mature B cell.

[0100] " in conjunction with the antibody of B cell surface marker " be with can destroy or consume the intravital B cell of mammal after the B cell surface marker combines and/or disturb the molecule of one or more B cell functions, for example, by weakening or preventing the humoral response that the B cell causes.Described antibody preferably can its processing of consumptive use the intravital B cell of mammal (promptly reducing the circulation b cell level).This consumption can realize by various mechanism, as the cytotoxicity (ADCC) of antibody dependent cellular mediation and/or depend on complement cytotoxicity (CDC), suppress B cell proliferation, and/or induce B cell death (for example passing through apoptosis).

[0101] " antagonist " refers to can neutralize, block, suppress, eliminate, reduce or disturb concrete or the active molecule of specified protein, described activity comprise with the part be example its with one or more receptors combine or with the receptor be example its with the combining of one or more parts.Antagonist comprises antibody and antigen-binding fragment thereof, protein, and peptide, glycoprotein, glycopeptide, glycolipid, polysaccharide, oligosaccharide, nucleic acid, biological organic molecule, peptide mimics, control sequence is transcribed and translated to pharmaceutical preparation and their metabolite, or the like.Antagonist also comprises proteinic micromolecular inhibitor, and thereby binding proteins specific matter is blocked itself and its bonded fusion rotein of target, acceptor molecule and derivant, proteinic antagonist variant, proteinic antisense molecule, RNA is fit, and at proteinic ribozyme.

[0102] " B cell surface marker antagonist " be with can destroy or consume the intravital B cell of mammal after the B cell surface marker combines and/or disturb the molecule of one or more B cell functions, for example, by weakening or preventing the humoral response that the B cell causes.Described antagonist preferably can its processing of consumptive use the intravital B cell of mammal (promptly reducing the circulation b cell level).This consumption can realize by various mechanism, as ADCC and/or CDC, inhibition B cell proliferation, and/or induces B cell death (for example passing through apoptosis).Antagonist in the scope of the invention comprises the antibody in conjunction with the B cell marking, synthetic or natural-sequence peptide, and fusion rotein and micromolecule antagonist can be chosen wantonly and be coupled to or be blended in cytotoxic agent.Example includes but not limited to, for example, and CD20 antibody, BR3 antibody (for example, WO0224909), BR3-Fc, or the like.

[0103] example of CD20 antibody comprises: " C2B8 " is called " Rituximab " now (U.S. Patent number 5736137); The 2B8 Muridae antibody of yttrium-[90]-labelling is called " Y2B8 " or " ibritumomab tiuxetan "

Can be available from IDEC drugmaker (IDECPharmaceuticals, Inc.) (U.S. Patent number 5736137; 1993/6/22 by ATCC with accession number HB11388 preservation, title is 2B8); Muridae IgG2a " B1 " is also referred to as " tositumomab ", optional using ¹³¹The I labelling with obtain " ¹³¹I-B1 " or " iodine I ¹³¹Tositumomab " antibody (BEXXAR ^TM), can be available from Corixa (also can referring to U.S. Patent number 5595721); Muridae monoclonal antibody " 1F5 " (Press etc., Blood 69 (2): 584-591 (1987) and variant thereof, (WO 2003/002607, Leung, S. to comprise " (framework-patched) that insert framework " or humanization 1F5; Be preserved in ATCC with HB-96450); Muridae 2H7 and chimeric 2H7 antibody (U.S. Patent number 5677180); Humanization 2H7 (referring to, for example, WO 04/056312; US20060024295); HUMAX-CD20 ^TMAntibody (gene steps C Compaq (Genmab), Denmark); The human monoclonal antibodies of listing among the WO2004/035607 (Teeling etc.); AME-133 ^TMAntibody (Applied Molecular Evolution In (Applied Molecular Evolution)); A20 antibody or its variant such as chimeric or humanization A20 antibody (being expressed as cA20, hA20 respectively) (US 2003/0219433, she this company of Miao's MultiCam (Immunomedics)); With monoclonal antibody L27 available from International Leukocyte Typing Workshop (international leukocyte typing operating room), G28-2,93-1B3, B-C1 or NU-B2 (Valentine etc., (McMichael compiles to be selected from LeukocyteTyping III (leukocyte typing III), the 440th page, Oxford University Press (Oxford University Press) (1987)).

[0104] " antirheumatic of alleviation disease " or the example of " DMARD " comprise hydroxyl clorotepine, sulfasalazine, methotrexate, leflunomide, Embrel, infliximab (adding oral and subcutaneous methotrexate), azathioprine, Beracilline, gold (oral), gold (intramuscular), minocycline, ciclosporin, SP immunoadsorption, comprise its salt and derivant, or the like.

[0105] " CTLA4 " expresses and participates in the downward modulation immunne response on the activated T lymphocyte.Other titles of CTLA4 comprise the cytotoxic T cell antigen 4 in the document, cytotoxic T cell associated protein 4, cell differentiation antigens c D152, relevant granule serine protease 4 with cytotoxic T cell.

[0106] many other terms have also carried out definition in the text or have characterized.

Detailed Description Of The Invention

[0107] CD4 mainly goes up the surface glycoprotein of expressing at T lymphocyte pedigree cell (comprising most of thymocyte cell and periphery T cell subclass).Some non-lymphocyte is also expressed low-level CD4, although the functional meaning that this divergent cell distributes is also unknown.On mature T cells, CD4 interacts by the MHC II quasi-molecule with the antigen-presenting cell expression and brings into play the other function of common recognition.CD4+T cell main composition is regulated the function accessory cell subclass of T and B cell during the T-of virus, antibacterial, fungus and parasitic infection dependency is replied.

[0108] in the pathogenesis of autoimmune disease when destroyed (especially when to the toleration of autoantigen), thereby the CD4+T cell can cause inflammatory reaction to cause joint and disorganization.For example,, produce antibody, inflammatory cytokine and medium, and help these processes by activating killer cell by raising the inflammatory cell of hematopoietic lineage.

[0109] the CD4+T cell is relevant with the pathogenesis of lupus.For example, the CD4+T cell is present in the glomerulonephritis position.It is reported that SLE patient's CD4+T cell is to the antigen overrespond and at external tolerance apoptosis.Can support the B cell to produce the autoantigen specific C D4+T cell of autoantibody (producing effect/memory CD4+ cell of IFN-γ) is present among the SLE patient.In addition, observe between MHC II level allele and the SLE risk relevant strongly.

[0110] similarly, the CD4+T cell is relevant with the pathogenesis of MS.For example, the CD4+ helper T cell participates in the pathogenesis of MS, corresponding laboratory model, experimental allergic encephalomyelitis (EAE), and the laboratory animal demonstration of having eliminated the T cell can not produce the EAE (USPN4695459 of Steinman etc., be entitled as " Method for the treatment of autoimmune diseases that are mediated byLeu3/CD4 phenotype T cells (method of the autoimmune disease that treatment Leu3/CD4 phenotype T is cell-mediated) ", Traugott etc., (1983) " Multiple sclerosis:distribution of T cell subsetswithin active chronic lesions (multiple sclerosis: the distribution of T cell subtype in the activeness chronic injury) " Science 219:308-310, Arnason etc., (1962) " Role of the thymus in immunereaction in rats:II.Suppressive effect of thymectomy at birth on reactions ofdelayed (cellular) hypersensitivity and the circulating small lymphocyte (effect of thymus in rat immunity reaction: the thymectomy that carries out during the II. birth is to (cell) allergy that postpones and the inhibitory action of circulation small lymphocyte) " J Exp Med 116:177-186, and Gonatas and Howard (1974) " Inhibition of experimental allergic encephalomyelitis in ratsseverely depleted of T cells (suppressor T cell seriously consumes the rat experimental allergic encephalitis) " Science 186:839-841).CD4+ and CD8+T cell are found in the MS damage; Known the two all produces inflammatory cytokine, although they are not clear for pathogenetic Relative Contribution.The frequency of observing myelin specific C D4+ cell in the MS blood samples of patients has improved 4 times.It is believed that some drug moieties present use or that be used for the treatment of MS in the future probably work to the effect of T cell by them; For example,

(natalizumab, α-4 alpha 2 integrin antibodies),

(alemtuzumab, CD52 antibody), and daclizumab (IL-2R Alpha antibodies).In addition, the I level allele (2 times) that the increase of MS risk and MHCII level allele (3.6 times) and degree are lower is relevant.

[0111] on the one hand, the invention provides by other combination of compounds that give non-expendable CD4 antibody and be used for treating lupus clinically or experimentally and treat lupus, comprise the method for SLE and lupus nephritis.Another aspect of the present invention provides by the non-expendable CD4 antibody that causes renal function improvement and/or albuminuria or activeness urinary sediment to alleviate and treats lupus nephritis, comprises the method that arrives terminal illness mid-term.A further aspect of the present invention provides the method for the treatment of MS with the non-expendable CD4 antibody of other chemical compounds combinations that are used for treating MS clinically or experimentally by choosing wantonly.The present invention provides in addition on the other hand by giving usually to treat with the non-expendable CD4 antibody of other chemical compounds combinations that are used for treating autoimmune disease clinically or experimentally suffers from autoimmune disease such as rheumatoid arthritis, asthma, psoriasis, inflammatory bowel (for example, Crohn disease and ulcerative colitis) and the graft receptor of xerodermosteosis or the method for object.

CD4 antibody

[0112] many expendables and non-expendable CD4 antibody have been described.Reported that also these antibody are inducing the application aspect the antigen toleration of (comprising autoantigen).Referring to, for example, USPN4695459; The USPN 6056956 of Cobbold and Waldmann is entitled as " Non-depleting anti-CD4monoclonal antibodies and tolerance induction (non-expendable resisting-CD4 monoclonal antibody and tolerance-induced) "; The USPN 5690933 of Cobbold and Waldmann is entitled as " Monoclonal antibodiesfor inducing tolerance (being used to induce the monoclonal antibody of patience) "; The European patent application published 0240344 of Cobbold etc. is entitled as " Monoclonal antibodies and their use (monoclonal antibody and application thereof) "; The USPN 6136310 of Hanna etc. is entitled as " Recombinant anti-CD4 antibodies forhuman therapy (the reorganization anti-CD 4 antibodies that is used for the human treatment) "; The USPN5756096 of Newman etc. is entitled as " Recombinant antibodies for human therapy (recombinant antibodies that is used for the human treatment) "; The USPN 5750105 of Newman etc. is entitled as " Recombinant antibodies for humantherapy (recombinant antibodies that is used for the human treatment) "; The USPN 4381295 of Kung and Goldstein is entitled as " Monoclonal antibody to human helper T cells and methods of preparingsame (monoclonal antibody of people's helper T cell and preparation method thereof) "; Waldmann (1989) " Manipulation of T-cell responses with monoclonal antibodies (monoclonal antibody is to the manipulation of t cell responses) " Ann Rev Immunol 7:407-44; And Wofsy and Seaman (1987) " Reversal of advanced murine lupus in NZB/NZW F1mice by treatment withmonoclonal antibody to L3T4 (reverse the senior Muridae lupus of NZB/NZW F1 mice with the L3T4 mab treatment " J Immunol 138:3247-53.Specifically, non-expendable CD4 antibody and and the application in inducing tolerance be described in the U.S. Patent Application Publication No. 2003/0108518 of Frewin etc., the U.S. Patent Application Publication No. 2003/0219403 that is entitled as " TRX1 antibody and uses therefor (TRX1 antibody and uses thereof) " and Frewin etc., be entitled as " Compositions and methodsof tolerizing a primate to an antigen (making primate tolerate antigenic compositions and method) ", include this paper separately by reference in.

[0113] the exemplary non-expendable CD4 antibody that is applicable to described method comprises TRX1 antibody, be described in the U.S. Patent Application Publication No. 2003/0108518 of Frewin etc., be entitled as the U.S. Patent Application Publication No. 2003/0219403 of " TRX1 antibody and uses thereof " and Frewin etc., be entitled as " making primate tolerate antigenic compositions and method ".These antibody are humanized antibodies, and it comprises the light chain of constant region, people's antibody of modified people's antibody and heavy chain framework region and derived from the light chain and the heavy chain CDR of mouse monoclonal antibody.

[0114] therefore, in a class embodiment, non-expendable CD4 antibody is as the TRX1 antibody shown in arbitrary among Fig. 1-4.This antibody can have heavy chain amino acid sequence shown in light-chain amino acid sequence shown in heavy chain amino acid sequence shown in light-chain amino acid sequence shown in heavy chain amino acid sequence, the SEQ ID NO:15 shown in light-chain amino acid sequence shown in heavy chain amino acid sequence, the SEQ ID NO:9 shown in light-chain amino acid sequence shown in the SEQ ID NO:3 and the SEQ IDNO:6 and the SEQ ID NO:12 and the SEQ ID NO:18 or the SEQ ID NO:21 and the SEQ ID NO:24.In the embodiment of correlation type, described antibody comprises the CD4 binding fragment of certain antibody, and this antibody comprises heavy chain amino acid sequence shown in light-chain amino acid sequence shown in heavy chain amino acid sequence shown in light-chain amino acid sequence shown in heavy chain amino acid sequence, the SEQ ID NO:15 shown in light-chain amino acid sequence shown in heavy chain amino acid sequence, the SEQ ID NO:9 shown in light-chain amino acid sequence shown in the SEQ ID NO:3 and the SEQ ID NO:6 and the SEQ ID NO:12 and the SEQ ID NO:18 or the SEQ ID NO:21 and the SEQ ID NO:24.

[0115] antibody that comprises the one or more CDR of TRX1 antibody also can be used for this method.Therefore, in a class embodiment, non-expendable CD4 antibody comprises CDR1 (SEQ IDNO:25), CDR2 (SEQ ID NO:26) or the preferred CDR3 (SEQ ID NO:27) of light chain shown in Figure 1A.Optional CDR1, CDR2 and the CDR3 (SEQ ID NO:25-27) that comprises light chain shown in Figure 1A of described antibody.Similarly, in a class embodiment, described antibody comprises CDR1 (SEQ ID NO:28), CDR2 (SEQ ID NO:29) or the preferred CDR3 (SEQ ID NO:30) of heavy chain shown in Fig. 1 D.Optional CDR1, CDR2 and the CDR3 (SEQ ID NO:28-30) that comprises heavy chain shown in Fig. 1 D of described antibody.In a class embodiment, described antibody comprises CDR1, CDR2 and the CDR3 (SEQ ID NO:25-30) of heavy chain shown in CDR1, the CDR2 of light chain shown in Figure 1A and CDR3 and Fig. 1 D.Optional FR1, FR2 and/or the FR3 that also comprises light chain shown in Figure 1A, Fig. 2 A, Fig. 3 A or Fig. 4 A of this antibody, and/or FR1, FR2, FR3 and/or the FR4 of heavy chain shown in Fig. 1 D, Fig. 2 D, Fig. 3 D or Fig. 4 D.

[0116] other exemplary antibodies include, but not limited to and the antibody of the identical epi-position of TRX1 antibodies (for example, as the antibody shown in arbitrary among Fig. 1-4).

[0117] when described to as if man-hour, described antibody is humanized antibody or people's antibody preferably.Obviously, when the treatment non-human mammal, choose wantonly and transform described antibody, for example framework and constant region sequence by mixing suitable species mammalian immune globulin to be applicable to this animal.This antibody is optional to be monoclonal antibody, complete antibody, antibody fragment and/or natural antibody.

[0118] this antibody is optional has the effector function that weakens, and for example, compares with the human IgG1, thereby reduces the Cytotoxic ability that it induces complement activation and/or antibody dependent cellular mediation.For example, this antibody and Fc receptor combines reduction (or not having).Similarly, this antibody can have nonglycosylated Fc part.This antibody is optional to be can be in conjunction with anti--CD4 variant antibody of FcRN.

The treatment of lupus

[0119] on the one hand, the invention provides the method that the anti--non-expendable antibody of CD4 by treating effective dose and/or second medicament are treated the lupus of mammalian object such as human subjects.The lupus of institute's treatment target is systemic lupus erythematosus (sle) (SLE), lupus erythematosus,cutaneous (CLE) or lupus nephritis normally, but also can be other forms of lupus, as the outer lupus of kidney, encephalitis lupus, department of pediatrics lupus, non-kidney lupus, discoid lupus or alopecia lupus.The lupus for the treatment of can be early stage, mid-term or terminal illness in when beginning treatment.In the embodiment of treatment lupus nephritis, described lupus nephritis can be any in the I-VI level.For example, the lupus that treat can be film hypertrophy lupus nephritis (II level) or film lupus nephritis (V level).Lupus normally is hypertrophy lupus nephritis (III level or an IV level), and therapeutic purposes are to make that albuminuria alleviates, the activeness urinary sediment alleviates and make renal function normal or stable.For example, albuminuria (is measured with means known in the art, for example, twenty-four-hour urine protein is measured, use scale (dip stick) or other conventional analyses, for example this paper embodiment 1 is described) can reduce at least 25% or at least 50%, or even at least 75% or at least 90%, perhaps albuminuria can be brought down below 1g/ days or be lower than 500mg/ days.Another example is, the urine protein of object can reduce at least 25% or at least 50% with the ratio of kreatinin, and perhaps this ratio can be brought down below 1 or be lower than 0.5.Similarly, the activeness urinary sediment (is monitored by means known in the art, for example by microexamination) can reduce at least 25%, at least 50%, at least 75% or at least 90%, or after the treatment beginning, only keep inactivity urinary sediment (be lower than 10 Red blood corpuscles/high power field and do not exist erythrocyte tubular, preferably be lower than 5 Red blood corpuscles/high power field susceptible of proof this point).On the one hand, when treatment during lupus nephritis, demonstrate with described object behind the described combination begin treatment that albuminuria alleviates and/or the activeness urinary sediment alleviates.For example, protein concentration in the object urine can be reduced to below 75% of protein concentration in the object urine before the described combination begin treatment, below 50%, below 25%, or below 10%, or be brought down below 1g/ days or be lower than 500mg/ days, and/or the activeness urinary sediment can reduce at least 25%, at least 50%, at least 75% or at least 90%, inactivity urinary sediment (for example, be lower than 10, preferably be lower than 5 Red blood corpuscles/high power field) is perhaps only arranged after treatment beginning.

In the conventional embodiment of [0120] one class, non-expendable CD4 antibody and at least a second combination of compounds that will treat effective dose in the method give object with the treatment lupus.Described non-expendable CD4 antibody can be as herein described any.Described second chemical compound is the chemical compound that is commonly used to treat lupus, for example, and the standard substance of nursing or experimental treatment.The second exemplary chemical compound includes, but not limited to cytotoxic agent; Immunosuppressant; Antimalarial such as oxychloroquine, chloroquine, or quinacrine; Chemotherapeutics; Cytokine antagonist or antibody; Somatomedin; Hormone (for example, Hormone Replacement Therapy); Anti--hormonotherapy; Integrin antagonist or antibody, for example, α 4-alpha 2 integrin antibodies or antagonist; B cell surface marker antagonist; The antibody of B cell surface marker (for example, CD20 antibody, for example, Rituximab is also referred to as

The antibody of CD5, CD28 or CD40 or blocker; 17-hydroxy-11-dehydrocorticosterone, for example, methylprednisolone, prednisone such as low dosage prednisone, dexamethasone, or glucocorticoid (glucorticoid) for example, through joint injection, comprise the treatment of general 17-hydroxy-11-dehydrocorticosterone; DMARD; Or above-mentioned any combination, or the like.Also can be referring to U.S. Patent Application Publication 2006/0024295 and 2003/0219403.

[0121] in a class embodiment, above-mentioned second chemical compound is selected from, for example, and cyclophosphamide, mycophenolate, CTLA4-Ig and BR3-Fc.Cyclophosphamide is that commodity are by name Mycophenolate is also referred to as

MMF or 2-morpholino ethyl (E)-6-(1,3-dihydro-4-hydroxyl-6-methoxyl group-7-methyl-3-oxygen-5-isobenzofuran-base)-4-methyl-4-hexene acid esters (hexenoate).CTLA4-Ig is the ectodomain that is connected in the people's cytotoxic T-lymphocyte-antigen 4 (CTLA-4) of modified human normal immunoglobulin Fc part, for example, and A Bate match general (abatacept) (Shi Guibao company during Bristol-U.S. ) or RG2077 (genome company (RepliGen) in next general).Exemplary α 4-alpha 2 integrin antibodies be natalizumab (

).The solubility antagonist BR3-Fc of BAFF be a kind of people of comprising BR3 ectodomain (the BAFF receptor of on the B cell, finding) and human IgG1 Fc fusion rotein (referring to, for example, Vugmeyster etc., (2006) American Journal of Pathology 168:476-489 and Kayagaki etc., (2002) Immunity 10:515-524).Can choose wantonly in this combination comprise the third, the 4th kind (or the like) chemical compound; An example is that 17-hydroxy-11-dehydrocorticosterone such as methylprednisolone and/or prednisone can give with CD4 antibody and cyclophosphamide.

In [0122] one embodiment, never use medicine before the object,, treated lupus and/or never treated before with anti-CD 4 antibodies as immunosuppressant.In another embodiment, healed with medicine before object lupus and/or treated with anti-CD 4 antibodies before.In further embodiment, object is not suffered from rheumatoid arthritis.In further embodiment, object is not suffered from multiple sclerosis again.Again in other embodiments, object is not suffered from the autoimmune disease outside the lupus." autoimmune disease " represent in the text by the tissue of intrasubject or organ or altogether separator cause and at this tissue or organ or the disease or the disease of separator altogether or its form of expression or the symptom that causes thus.In one embodiment, it refers to owing to the B cell produces the symptom that causes or aggravate with normal bodily tissue and antigen reactive antibody.In other embodiments, described autoimmune disease relates to the secretion of specific autoantibody of the epi-position of autoantigen (for example nuclear antigen).

In [0123] one embodiment, before described combination begin treatment, described object demonstrates albuminuria, has improved this albuminuria by treatment.For example, before the treatment beginning, the albuminuria that object demonstrates is higher than 500mg/ days, is higher than 1000mg/ days, is higher than 2000mg/ days or is higher than 3500mg/ days; After the treatment beginning, albuminuria can reduce at least 25% or at least 50%, and perhaps even at least 75% or at least 90%, perhaps albuminuria can be brought down below 1g/ days or be lower than 500mg/ days, for example, records by the measurement of twenty-four-hour urine protein.

[0124] protein also can be monitored similarly with the reduction of the ratio of kreatinin.Urine protein and creatinine levels can be measured by means known in the art, for example, and the urine protein by measuring urine sample at random and the ratio of kreatinin.In one embodiment, before described combination begin treatment, the ratio that described object demonstrates protein and kreatinin is higher than 0.5, be higher than 1 or be higher than 2; After the treatment beginning, protein may be reduced to the ratio of kreatinin, for example, is lower than 1 (for example, to the treatment partial response time) or be lower than 0.5 (when for example, responding fully).After the treatment beginning, compare with the value before the treatment, protein can reduce at least 25% or at least 50% with the ratio of kreatinin.In one embodiment, before described combination begin treatment, described object demonstrates the albuminuria of nephropathy scope, and promptly protein is higher than 3 with the ratio of kreatinin; After the treatment beginning, protein is brought down below 3 with the ratio of kreatinin, perhaps optionally reduces at least 25% or at least 50% or be brought down below 2 or be lower than 1.

[0125] for example, can estimate with this combined therapy lupus, comprise the reaction of lupus nephritis by monitoring complement level, autoantibody level and/or overall disease activity.For example, the normalization of complement level (for example, C3, C4 and CH50) can show and treats successfully.Similarly, after the treatment beginning, autoantibody for example can reduce at least 25%, at least 50% or at least 75% as the level of anti--double-stranded-DNA antibody, ANA and anti--Clq.Also can be observed the improvement of kidney biopsy, thereby show and treat successfully.

[0126] randomly, before described combination begin treatment, described object demonstrates nephrotic syndrome.Can pass through means known in the art nephropathy diagnosis syndrome.Some symptom, symptom or other signs can be used to the nephropathy diagnosis syndrome, be higher than 3.5g/ days comprising twenty-four-hour urine protein, protein is higher than 3 with the ratio of kreatinin, hypoalbuminemia (albumin level in the blood is low), especially the edema (swelling) around eye, foot and the hands, and/or hypercholesterolemia (cholesterol levels height in the blood).The invention is not restricted to these symptom, symptom or other signs of nephrotic syndrome.Can choose the alleviate kidney disease syndrome wantonly with this combined therapy.For example, the described object in treatment beginning back is optional to be demonstrated albuminuria and is relieved to and is lower than 3.5g/ days, for example, is lower than 3g/ days, be lower than 2g/ days, be lower than 1g/ days, or even be lower than 0.5g/ days, and/or the ratio of treatment beginning back protein and kreatinin is brought down below 3, for example, be lower than 2, be lower than 1, or even be lower than 0.5.

[0127] for object considerable benefit is arranged with this combined therapy object, for example, can alleviate adverse side effect.For example, with the amount of required second chemical compound (for example, cyclophosphamide) of non-expendable CD4 antibody combined therapy can be significantly less than single with this second compounds for treating with the required amount of relief of symptoms.For example, cyclophosphamide can produce serious side effects; Therefore press for less medicine and finish treatment.

[0128] on the one hand, this method comprises with non-expendable CD4 antibody and the second compounds for treating object and to continue then to disappear to keep with non-expendable CD4 antibody (not making up with second chemical compound) treatment target with mitigation symptoms.For example, the combined therapy object of available non-expendable CD4 antibody and cyclophosphamide, mycophenolate or CTLA4-Ig is with mitigation symptoms, list disappears to keep with non-expendable CD4 antibody (that is, not making up with cyclophosphamide, mycophenolate or CTLA4-Ig) treatment then.Make the method for the second minimum chemical compound of object contact also can reduce side effect.In other embodiments,, disappear to keep with second chemical compound except that non-expendable CD4 antibody or one or more other chemical compounds continuation treatment targets then with mitigation symptoms with non-expendable CD4 antibody and the second compounds for treating object.

[0129] another kind of conventional embodiment also provides for example method of people's lupus nephritis of treatment mammalian object.In the method, the non-expendable CD4 antibody with the treatment effective dose gives object.Behind described antibody begin treatment, to compare with albuminuria and/or activeness urinary sediment level that object before the treatment beginning demonstrates, described object demonstrates renal function to be improved, and albuminuria alleviates, and/or the activeness urinary sediment alleviates.For example, albuminuria can reduce at least 25%, at least 50%, at least 75% or at least 90%, and perhaps albuminuria can be brought down below 1g/ days or be lower than 500mg/ days; Urine protein can reduce at least 25% or at least 50% with the ratio of kreatinin, and perhaps this ratio can be brought down below 1 or be lower than 0.5; And/or the activeness urinary sediment can reduce at least 25%, at least 50%, at least 75% or at least 90%, perhaps after the treatment beginning inactivity urinary sediment only arranged.Described non-expendable CD4 antibody can be as herein described any.

In [0130] one embodiment, lupus nephritis and/or never treated with anti-CD 4 antibodies before never healed with medicine before the object.In other embodiments, healed with medicine before object lupus nephritis and/or treated with anti-CD 4 antibodies before.In other embodiments, non-expendable anti-CD 4 antibodies of the present invention is the unique medicine of object with the treatment lupus nephritis that give.In other embodiments, non-expendable CD4 antibody of the present invention is to be used for one of medicine for the treatment of lupus nephritis.In further embodiment, described object is not suffered from rheumatoid arthritis.In further embodiment, described object is not suffered from multiple sclerosis again.Again in other embodiments, described object is not suffered from the autoimmune disease except that lupus and/or lupus nephritis.

[0131] in a class embodiment, this method comprises and gives at least a second chemical compound, any in the chemical compound of CD4 antibody combination as described herein and non-expendable.For example, cyclophosphamide, mycophenolate, CTLA4-Ig or α 4-alpha 2 integrin antibodies can give object with non-expendable CD4 antibody combination.Can choose wantonly in this combination comprise the third, chemical compound such as the 4th kind; For example, 17-hydroxy-11-dehydrocorticosterone such as methylprednisolone and/or prednisone can give with CD4 antibody and cyclophosphamide.

In [0132] one embodiment, before beginning with non-expendable CD4 Antybody therapy, described object demonstrates albuminuria, reduces with this albuminuria behind the non-expendable CD4 antibody begin treatment.For example, before the treatment beginning, the albuminuria that object demonstrates is higher than 500mg/ days, is higher than 1000mg/ days, is higher than 2000mg/ days or is higher than 3500mg/ days; After the treatment beginning, albuminuria can reduce at least 25%, at least 50%, at least 75% or at least 90%, and perhaps albuminuria can be brought down below 1g/ days or be lower than 500mg/ days.Can monitor the reduction of protein and the ratio of kreatinin similarly.In one embodiment, before the treatment beginning, the ratio that described object demonstrates protein and kreatinin is higher than 0.5, be higher than 1 or be higher than 2; After the treatment beginning, protein can reduce at least 25% or at least 50% with the ratio of kreatinin, or is brought down below 1 or be lower than 0.5.In one embodiment, before described combination begin treatment, described object demonstrates the albuminuria of nephropathy scope, and promptly protein is higher than 3 with the ratio of kreatinin; After the treatment beginning, protein is brought down below 3 with the ratio of kreatinin, perhaps optionally reduces at least 25% or at least 50% or be brought down below 2 or be lower than 1.Randomly, before the treatment beginning, described object demonstrates nephrotic syndrome.Nephrotic syndrome can be chosen wantonly by treatment and alleviate.For example, treatment beginning back object can be chosen wantonly and demonstrate albuminuria and alleviate to being lower than 3.5g/ days, for example, is lower than 3g/ days, is lower than 2g/ days, is lower than 1g/ days, perhaps even be lower than 1g/ days or be lower than 0.5g/ days.

The treatment multiple sclerosis

[0133] multiple sclerosis (MS) is a kind of people central nervous system's (CNS) inflammatory and a demyelination degenerative disease.It is a kind of worldwide disease, has about 300,000 people to suffer from this disease in the U.S.; It mainly occurs among the young adult, about 70%-80% patient's age of onset 20-40 between year (Anderson etc., Ann Neurology 31 (3): 333-6 (1992); Noonan etc., Neurology 58:136-8 (2002)).Based on its clinical course, the pathological analysis of nuclear magnetic resonance (MRI) scanning evaluation and biopsy and postmortem material, MS is a kind of different substantiality disease (Lucchinetti etc., Ann Neurol 47:707-17 (2000)).This disease has multiple performance, comprises spinal cord, brain stem, cranial nerve, cerebellum, brain and cognitive syndrome.Most of MS patients have progressive disability, especially in following 25 years.The MS needs of patients walking stick of half is walked in 15 years after disease takes place.MS is the main cause that causes young and middle aged neurologic disability, does not also know useful Therapeutic Method up to past 10 years.Owing to do not have specificity clinical discovery, MS to be difficult to diagnosis, therefore need the diagnostic criteria of exploitation highly structural, wherein comprise some technological progresses, comprise MRI scanning, bring out current potential and cerebrospinal fluid (CSF) research.All diagnostic criterias are depended on the General Principle of the interior dispersion damage of central white matter that different time takes place and can not be explained (McDonald etc., Ann Neurol 50:121-7 (2001)) by other etiology such as infection, angiopathy or other autoimmune conditions.MS has four kinds of disease patterns: recurrent-intermittence (relapsing-remitting) MS (RRMS; The case of 80%-85% during morbidity), carrying out property of constitutional MS (PPMS; 10%-15% during morbidity), carrying out property recurrent MS (PRMS; During morbidity 5%); With carrying out property of Secondary cases MS (SPMS) (Kremenchutzky etc., Brain 122 (the 10th part): 1941-50 (1999); Confavreux etc., N Eng1 J Med 343 (20): 1430-8 (2000)).Estimate that 50% RRMS patient will develop into SPMS in 10 years, and have nearly that 90% RRMS patient will finally develop into SPMS (Weinshenker etc., Brain 112 (part 1): 133-46 (1989)).

[0134] the present invention includes the method for the multiple sclerosis of treatment mammalian object such as human subjects.On the one hand, this method comprises the non-expendable CD4 antibody that gives described object treatment effective dose.Described non-expendable CD4 antibody can be as herein described any.In other respects, this method comprises non-expendable CD4 antibody and at least a second combination of compounds that gives described object treatment effective dose.Equally, described non-expendable CD4 antibody can be as herein described any.

[0135] described second chemical compound is commonly used to treat MS, for example, and the standard substance of nursing or experimental treatment.Exemplary second chemical compound includes, but not limited to cytotoxic agent; Immunosuppressant (for example, cyclophosphamide); B cell surface marker antagonist; B cell surface marker antibody; CD20 antibody, for example, Rituximab is seen US 20060051345); The antibody of CD5, CD28 or CD40 or blocker; Antibody or the antagonist such as the natalizumab of 17-hydroxy-11-dehydrocorticosterone (for example, prednisone), CTLA4-Ig, α 4-integrin

Mycophenolate, inhibin, the antibody of LFA-1 or CD-11a or blocker are (referring to the U.S. Patent Application Publication No. 20050281817 of Jardieu etc., be entitled as " Method for treating multiplesclerosis (method of treatment multiple sclerosis) ", il-1 2 antibody, interferon-(for example, interferon beta-1a as

Or

Or interferon beta-1b as

), the acetic acid glatiramer

CD52 antibody such as alemtuzumab

Interleukin-2-receptor antibody such as daclizumab Interleukin-2 receptor alpha subunit antibody), or the like.

[0136] in a class embodiment, this method comprises with non-expendable CD4 antibody and the second compounds for treating object to be used non-expendable CD4 antibody (with the combination of second chemical compound) to continue treatment target then and disappears to keep with mitigation symptoms.For example, object can singly disappear to keep with non-expendable CD4 Antybody therapy then with the combined therapy of non-expendable CD4 antibody and acetic acid glatiramer.In other embodiments, object can be with non-expendable CD4 antibody and second compounds for treating with mitigation symptoms, and the chemical compound that is commonly used to treat MS with second chemical compound except that non-expendable CD4 antibody or one or more continues to treat then.

In [0137] one embodiment, object is never crossed multiple sclerosis with medicine such as immunosuppressant treatment before and/or was never treated with anti-CD 4 antibodies before.In other embodiments, healed with medicine before object multiple sclerosis and/or treated with anti-CD 4 antibodies before.

[0138] common, described object is to fit lattice for the treatment of multiple sclerosis, and is promptly described to liking the MS object.For the purpose of this paper, this MS experiences, once experiences or may experience the object of one or more symptom, symptom or other signs of multiple sclerosis to liking; Diagnosed out the object of multiple sclerosis, for example no matter be (" de novo " MS) that newly diagnose out, diagnose out recurrence again before or worsen, diagnose out before and disappear, or the like; And/or the object of multiple sclerosis risk arranged.Evaluation suffers from multiple sclerosis or has the man-hour of ill risk can choose wantonly by the positive b cell level rising of CD20 in serum, cerebrospinal fluid (CSF) and/or the MS damage, and/or adopts the test that detects autoantibody to screen qualitative and preferred qualitative assessment.The exemplary relevant autoantibody of this and multiple sclerosis comprises anti--myelin basic protein (MBP), resists-myelin oligodendrocyte glycoprotein (MOG), resists-ganglioside and/or anti--neurofilament antibody.Can in serum, cerebrospinal fluid (CSF) and/or the MS damage of object, detect this autoantibody." rising " autoantibody or b cell level are meant that in the text the level of this autoantibody or B cell obviously surpasses the level of the individuality of not suffering from MS.

[0139] in the literary composition, the MS that treat comprises carrying out property of constitutional multiple sclerosis (PPMS), recurrent-intermittent multiple sclerosis (RRMS), carrying out property of Secondary cases multiple sclerosis (SPMS) and carrying out property recurrent multiple sclerosis (PRMS).Described MS can be early stage, mid-term or terminal illness when when beginning treatment.Statement " treatment effective dose " is meant that the consumption of antibody (the perhaps antibody and at least the second combination of compounds) can effectively prevent, alleviates or treat multiple sclerosis when being used for the MS treatment.This effective dose will improve symptom, symptom or other signs of MS usually, as reduce that relapse rate, prevention are disabled, the number and/or volume, the regularly 25-foot walking (timed 25-foot walk) of improvement that reduce brain MRI damage, prolong the progression of disease time and (for example adopt disability status scale (Expanded Disability Status Scale, EDSS)) of expansion or the like.On the one hand, the demyelination of treatment target is lowered.

[0140] " carrying out property of constitutional multiple sclerosis " or " PPMS's " is characterized as disease from its outbreak progress progressively, at all without any synergetic recurrence with disappear.May there be the disease activity stage of stable development, had situation then better and relatively poor several days (or a few week).The difference of PPMS and RRMS and SPMS is, it is early stage that it betides 30 years old late period or 40 years old usually, and masculinity and femininity is ill equally easily, and initial disease activity appears at spinal cord but not in the brain usually.PPMS can be moved to brain usually, but compares PRMS or SPMS unlikely damages brain region; For example, PPMS patient unlikely produces cognitive question.PPMS is the hypotype of MS, least may show inflammatory (gadolinium enhancing) damage in MRI scanning.This carrying out property of sick constitutional form accounts for the 10-15% of all multiple sclerosis patients.Can be according to McDonald etc., the standard definition PPMS of Ann Neurol 50:121-7 (2001).The PPMS object of this paper treatment normally might or be diagnosed as the object of PPMS.

[0141] " recurrent-intermittent multiple sclerosis " or " RRMS " be characterized as recurrence (being also referred to as deterioration), during new symptom or old symptom may occur and reappear or worsen.After the recurrence is paracmasis, during the patient can from the defective of recurrence, recover wholly or in part.Recurrence can last for days, several weeks or several months, and recover can be slowly and gradually or almost be moment.Most MS patients at first are diagnosed as RRMS.This usually occur in they 20 or 30 years old the time, although early much many or late to diagnose also be known.The women who suffers from this MS hypotype is male's a twice.During the recurrence, in myelin-central nervous system (CNS) white matter zone around the protectiveness insulation sheaths of nerve fiber (neuron)-can be in inflammatory reaction by the immune system injury of health self.This causes many neurological symptoms result that obviously depend on the CNS damage field and change.After the recurrence, inflammatory response disappears immediately, and a kind of neurogliocyte of specific type (being called oligodendrocyte) causes a kind of process that the myelin sheath around Remyelination-the make aixs cylinder may be repaired among the CNS.This just Remyelination may cause resolution of symptoms.About 50% RRMS patient is transformed into SPMS in morbidity in 10 years.After 30 years, this numeral rises to 90%.In any moment, this palindromia-form that disappears accounts for all MS patients' 55%.

[0142] " carrying out property of Secondary cases multiple sclerosis " or " SPMS's " is characterized as clinical neurology damage steady progress and is with or without synergetic recurrence and minimum disappear and steadily.Live through the RRMS phase before the SPMS patient, will continue 2-40 or longer this period.Any synergetic recurrence and disappearing reduces in time.Carry out the sexual stage outbreak from the Secondary cases of disease, compare the disabled beginning of RRMS stage and produce quickly, although this process also is very slowly in some individuality.After 10 years, 50% RRMS patient will develop into SPMS.During year, this numeral is increased to 90% at 25-30.Inflammatory damage among the SPMS forms level will be lower than RRMS, but the total load of disease can continue progress.In any moment, SPMS accounts for about 30% of multiple sclerosis patients.

[0143] " carrying out property recurrent multiple sclerosis " or " PRMS " be characterized as clinical neurology damage steady progress and with synergetic recurrence with disappear.After the recurrence is tangible recovery immediately, but symptom can run down between twice recurrence.PRMS influences about 5% of all multiple sclerosis patients.Some neurosurgeons think that PRMS is the version of PPMS.

Other treatment of conditions

[0144] non-expendable CD4 antibody comprises non-expendable CD4 antibody and one or more other combination of compounds, also can be used for disease and disease except that lupus or multiple sclerosis, for example, and CD4 ⁺The pathological condition that the T cell causes.Therefore, one aspect of the present invention provides the method for the disease of treatment mammalian object such as human subjects.This method comprises non-expendable CD4 antibody and at least a second combination of compounds that gives described object treatment effective dose.In one embodiment, described to as if the tissue grafts receptor, and the disease that will treat is transplant rejection or graft versus host disease.Other diseases of the enough said composition treatments of energy include, but not limited to autoimmune conditions or disease such as rheumatoid arthritis, asthma, psoriasis, inflammatory bowel (for example, Crohn disease or ulcerative colitis) and xerodermosteosis.

[0145] described non-expendable CD4 antibody can be above-mentioned any.It is to be used for treating described disease that this second chemical compound can be chosen wantonly, for example, and the standard substance of nursing or experimental treatment.Exemplary second chemical compound includes, but not limited to cytotoxic agent; Immunosuppressant (for example, cyclophosphamide); B cell surface marker antagonist; B cell surface marker antibody; CD20 antibody, for example, Rituximab is seen US20060051345); The antibody of CD5, CD28 or CD40 or blocker; Antibody or the antagonist such as the natalizumab of 17-hydroxy-11-dehydrocorticosterone (for example, prednisone), CTLA4-Ig, α 4-integrin

Mycophenolate, inhibin, the antibody of LFA-1 or CD-11a or blocker are (referring to the U.S. Patent Application Publication No. 20050281817by of Jardieu etc., be entitled as " method of treatment multiple sclerosis "), il-1 2 antibody, interferon-(for example, interferon beta-1a as

Or

Or interferon beta-1b as

The acetic acid glatiramer

, CD52 antibody such as alemtuzumab , interleukin-2-receptor antibody such as daclizumab (

Interleukin-2 receptor alpha subunit antibody), or the like.Other exemplary second chemical compounds are described in this article and/or are known in the art.Randomly, described second chemical compound is selected from cyclophosphamide, mycophenolate and CTLA4-Ig.

[0146] in a class embodiment, this method comprises with non-expendable CD4 antibody and the second compounds for treating object to be used non-expendable CD4 antibody (with the combination of second chemical compound) to continue treatment target then and disappears to keep with mitigation symptoms.In other embodiments, with mitigation symptoms, the chemical compound that is commonly used to treat described disease with second chemical compound or one or more continues treatment to described object then with non-expendable CD4 antibody and second compounds for treating.

In [0147] one embodiment, disease and/or never treated before as described in never crossing as immunosuppressant treatment before the object with anti-CD 4 antibodies with medicine.In other embodiments, healed with medicine before the object described disease and/or treated with anti-CD 4 antibodies before.

[0148] common, described object is to fit lattice for described treatment of conditions.For the purpose of this paper, this object that experiences liking, once experiences or may experience one or more symptom, symptom or other signs of described disease; Diagnosed out the object of described disease, for example no matter be newly diagnose out, diagnose out recurrence again before or worsen, diagnose out before and disappear, or the like; And/or the object of described disease risk arranged.For example, object for treatment transplant rejection or the suitable lattice of graft versus host disease can be will carry out tissue transplantation or accept this transplanting, and this object can be the object that experiences, once experiences or may experience one or more symptom, symptom or other signs of transplant rejection or graft versus host disease in the later case.The symptom of this disease and various autoimmune disease and disease and sign are well known in the art.

Production of antibodies and giving

[0149] the inventive method adopts the antibody in conjunction with CD4.On the one hand, described anti-CD 4 antibodies is non-expendable antibody.Therefore this paper will describe the method that produces this antibody.

[0150] T4 antigen that is used for producing or screen antibody can be, for example, the soluble form of CD4 as people CD4, or contains its part of required epi-position.Nucleic acid and the aminoacid sequence of people CD4 are shown in Figure 21.Perhaps, or in addition, the cell at cell surface expression CD4 can be used to produce or screening antibody.The other forms of CD4 that is used to produce antibody will be that those skilled in the art are conspicuous.

[0151] Xia Mian description relates to the illustrative preparation technology that is used for antibody of the present invention.Other information can be referring to the U.S. Patent Application Publication No. 2003/0108518 of Frewin etc., the U.S. Patent Application Publication No. 2003/0219403 that is entitled as " TRX1 antibody and uses thereof " and Frewin etc., be entitled as " making primate tolerate antigenic compositions and method ", for all purposes, comprise the method that produces non-expendable CD4 antibody such as TRX1 antibody, the both includes this paper by reference in full in.

Polyclonal antibody

[0152] polyclonal antibody is preferably by repeatedly subcutaneous or peritoneal injection related antigen and adjuvant produce from animal.Making related antigen is useful with have immunogenic protein in treating immune species as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor coupling, difunctionality reagent or derivating agent are adopted in coupling, for example, maleimide aminobenzoyl thiosuccimide ester (by the cysteine residues coupling), N-hydroxy-succinamide (by the lysine residue coupling), glutaraldehyde, succinic anhydrides, SOCl ₂Or R ' N=C=NR, wherein R is different alkyl with R '.

[0153] with antigen, immunogenic conjugate or derivant immune animal, for example the Freund's complete adjuvant of 100 μ g or 5 μ g protein or conjugate (being respectively applied for rabbit or mice) and 3 times of volumes is mixed during immunity is incorporated in a plurality of positions intradermal and injects this solution.Peptide or conjugate (preparing with Freund's complete adjuvant) with initial amount 1/5 to 1/10 after 1 month pass through a plurality of positions subcutaneous injection to the animal booster immunization.Gather animal blood after 7-14 days and measure the antibody titer of serum.The booster immunization animal is steady up to titre once more.Preferably, with the conjugate booster immunization animal that is coupled to different proteins and/or passes through the link coupled same antigen of cross-linking agent.Conjugate also can be used as the protein blend compound and prepares in the reconstitution cell culture.Simultaneously, agglutinant such as Alumen also are fit to be used for enhance immunity and reply.

Monoclonal antibody

[0154] monoclonal antibody is the antibody from the basic homogenizing of a group, and it is identical and/or in conjunction with same epi-position promptly to constitute each antibody of this colony, and except producing variant when the preparation monoclonal antibody, this variant exists with minimum usually.Therefore, modifier " monoclonal " shows that being characterized as of this antibody is not mixture that disperse or polyclonal antibody.

[0155] for example, monoclonal antibody can be by the earliest by Kohler etc., Nature, and the hybridoma method preparation that 256:495 (1975) describes perhaps can be passed through recombinant DNA manufactured (referring to, for example, U.S. Patent number 4816567).

[0156] in hybridoma method, mice or other suitable hosts animals such as hamster maybe can produce the lymphocyte of specificity in conjunction with the proteinic antibody that is used for immunity through immunity (as mentioned above) to cause to produce.Perhaps, can be at external immune lymphocyte.Adopt then and stablize fusion agent such as Polyethylene Glycol makes lymphocyte and myeloma cell merge formation hybridoma (Goding, Monoclonal Antibodies:Principlesand Practice (monoclonal antibody: principle with put into practice), 59-103 page or leaf (academic press, 1986)).

[0157] hybridoma that will so make is seeded in the proper culture medium and cultivates, and culture medium preferably contains the material of one or more parental generation myeloma cell that can suppress not merge growths or survival.For example, if parental generation myeloma cell lacks hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium of this hybridoma generally includes hypoxanthine, aminopterin, and thymidine (HAT culture medium), material wherein can stop the growth of HGPRT defective cell.

[0158] myeloma cell who can be used for preparing hybridoma is that those can effectively merge, can support the antibody produced cell selected stably high level produce antibody, and to the medullary cell of culture medium such as HAT culture medium sensitivity.Wherein, the non-limitative example of myeloma cell line comprises rat bone marrow tumour system, as (can happy from Maryland, USA Roc dimension (Rockville, American Type Culture Collection Md.USA) (AmericanType Culture Collection) obtains) derived from MOPC-21 and MPC-11 mouse tumor (can buy) and SP-2 or X63-Ag8-653 cell from Sol gram institute's cell distributing center (SalkInstitute Cell Distribution Center) in California, USA Santiago.Human myeloma and mice-people's allos myeloma cell line also is used for producing human monoclonal antibodies (Kozbor, J.Immunol., 133:3001 (1984); Brodeur etc., MonoclonalAntibody Production Techniques and Applications (technology and application that monoclonal antibody produces), 51-63 page or leaf, Marcel Dekker, Inc., New York, 1987)).

[0159] culture medium of check growth hybridoma produces the situation at antigenic monoclonal antibody.Can by immunoprecipitation or by external in conjunction with test, measure the binding specificity of the monoclonal antibody that hybridoma produces as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).For example, can analyze the binding affinity that (Munson etc., Anal.Biochem., 107:220 (1980)) measures monoclonal antibody by Scatchard.

[0160] identify can produce hybridoma with required specificity, affinity and/or active antibody after, can carry out sub-clone and cultivate (Goding the clone by limiting dilution assay by standard method, Monoclonal Antibodies:Principles and Practice (monoclonal antibody: principle with put into practice), 59-103 page or leaf (academic press, 1986)).The suitable culture medium that is used for this purpose comprises, for example, and D-MEM or RPMI-1640 culture medium.In addition, hybridoma can be cultivated in animal body as ascites tumour.

[0161], coagulates as A albumen-agarose by conventional immunoglobulin purification method

Sepharose, hydroxyapatite chromatography method, gel electrophoresis, dialysis or affinity chromatography can in the ascites, or be separated in the serum with the excretory monoclonal antibody of sub-clone suitably from cultivate base.

[0162] with conventional method (as, employing can with the bonded oligonucleotide probe of encoding gene specificity of Muridae heavy chain of antibody and light chain), the DNA with the coding monoclonal antibody of being not difficult separates and checks order.Hybridoma can be used as the useful source of this kind DNA.In case separate, this DNA can be inserted in the expression vector, dye then into host cell such as Bacillus coli cells, monkey COS cell, Chinese hamster ovary (CHO) cell, or do not produce in addition among the myeloma cell of immunoglobulin, to be implemented in synthetic monoclonal antibody in the recombinant host cell.Relevant in antibacterial the document of recombinant expressed antibody coding DNA comprise Skerra etc., Curr.Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol.Revs., 130:151-188 (1992).

[0163] in further embodiment, antibody or antibody fragment are separable from adopting McCafferty etc., Nature, the antibody phage library that the technology that 348:552-554 (1990) describes produces.Clackson etc., Nature, 352:624-628 (1991) and Marks etc., J.Mol.Biol., 222:581-597 (1991) have described the employing phage library respectively and how to have separated Mus and people's antibody.Publication has afterwards been described by chain reorganization and has been produced high-affinity (nM rank) people's antibody (Marks etc., Bio/Technology, 10:779-783 (1992)), and utilize combination infection and the interior reorganization of body to make up very large phage library (Waterhouse etc., Nuc.Acids.Res., 21:2265-2266 (1993)).Therefore, these technology are the feasible replacement methods of separating traditional monoclonal antibody hybridoma technology of monoclonal antibody.

[0164] DNA also can be modified, for example, and by the coded sequence replacement homology Muridae sequence (U.S. Patent number 4816567 of personnel selection heavy chain and light chain constant domain; Morrison etc., Proc.Natl.Acad.Sci.USA, 81:6851 (1984)), or by the part coded sequence covalent bond with whole immunoglobulin coding sequences or NIg polypeptide.

[0165] replaces the constant domain of certain antibody with this NIg polypeptide usually or replace the variable domains of an antigen binding site of certain antibody, producing a kind of chimeric bivalent antibody, this antibody contains one to have specific antigen binding site and synantigen is not had specific another antigen binding site certain antigen.

Humanized antibody

[0166] the humanized method of non-human antibody was described in this area.Preferably, humanized antibody has from one or more amino acid residue of non-human source introducing.These inhuman amino acid residues often are called " input " residue, and this residue is taken from " input " variable domains usually.Humanization in fact can be according to Winter and colleague's thereof method (Jones etc., Nature, 321:522-525 (1986); Riechmann etc., Nature, 332:323-327 (1988); Verhoeyen etc., Science, 239:1534-1536 (1988)) carry out, replace the corresponding sequence of people's antibody with the hypervariable region sequence.Therefore, such " humanization " antibody is chimeric antibody (U.S. Patent number 4816567), is wherein replaced by non-human animal's corresponding sequence less than complete people's variable domains basically.In fact, humanized antibody is people's antibody normally, and some of them hypervariable region residue and some possible FR residues are replaced by the residue at the similar position of rodent animal antibody.

[0167] selection that is used for preparing people's variable domains (light chain and heavy chain) of humanized antibody is crucial for reducing antigenicity.According to so-called " optimum matching (best-fit) " method, with the sequence of the complete library screening rodent animal antibody variable domains of known people's variable domains sequence.The human sequence approaching with the rodent sequence is people's framework region (FR) (Sims etc., J.Immunol., the 151:2296 (1993) of acceptable humanized antibody; Chothia etc., J.Mol.Biol., 196:901 (1987)).Another kind method adopts the specific framework region derived from the consensus sequence of everyone antibody of light chain and the specific subgroup of variable region of heavy chain.Identical framework can be used for some different humanized antibodies (Carter etc., Proc.Natl.Acad.Sci.USA, 89:4285 (1992); Presta etc., J.Immunol., 151:2623 (1993)).

[0168] importantly want humanized antibody to keep to antigenic high affinity and other favourable biological characteristics.In order to reach this target,, utilize the threedimensional model of parental generation and humanization sequence to prepare humanized antibody by the humanization product process of analyzing parental generation sequence and different concepts according to a kind of method.Three-dimensional immunoglobulin model can be buied and be familiar with by those skilled in the art usually.The computer program market of possible 3-d modelling structure of setting forth and demonstrating selected candidate's immunoglobulin sequences is on sale.Check that these demonstrations are analyzed residue may act in the function of candidate's immunoglobulin sequences, promptly analyze the residue that can influence immunoglobulin and its antigen binding capacity.Like this, obtain required antibody characteristic, increase as affinity to target antigen thereby can from receptor's sequence and list entries, select the FR residue and make up.In a word, direct and the most substantive participation of hypervariable region residue to the bonded influence of antigen.

People's antibody

[0169] substitutes as humanized, can produce people's antibody.For example, can produce transgenic animal (as mice) now, can produce through these animals of immunity inoculation does not have the people of endogenous immunoglobulin product antibody library.For example, the existing description said heavy chain of antibody bonding pad (J in mosaic and germ line mutation mice _H) the homozygosity disappearance of gene causes suppressing fully endogenous production of antibodies.With ethnic group is that the immunoglobulin gene array is transferred to and caused antigen to attack descendant's production of antibodies in this class germ line mutation mice.Referring to, for example, Jakobovits etc., Proc.Natl.Acad.Sci.USA, 90:2551 (1993); Jakobovits etc., Nature, 362:255-258 (1993); Bruggermann etc., Year in Immuno., 7:33 (1993); And U.S. Patent number 5591669,5589369 and 5545807.

[0170] in addition, can adopt display technique of bacteriophage (McCafferty etc., Nature 348:552-553 (1990)) the never immunoglobulin variable of immunity inoculation donor (V) district's gene library external generation people's antibody and antibody fragment.According to this technology, can will be cloned in the antibody V district gene frame in the big or parcel memebrane protein gene of filobactivirus such as M13 or fd, and be the functional antibodies fragment at the phage particle surface display.Because filamentous particle contains the single stranded DNA copy of phage genome, also caused showing the selection of encoding gene of the antibody of these characteristics based on the selection of antibody function characteristic.Thereby phage has been simulated the part characteristic of B cell.Phage display can carry out in a variety of forms; Summary is seen for example Johnson, KevinS. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993).Some sources of V constant gene segment C can be used for phage display.Clackson etc., Nature, 352:624-628 (1991) separates the multiformity array that obtains anti-azolactone antibody from the little combinatorial library at random of V gene that the immune mouse spleen obtains.Basically according to Marks etc., J.Mol.Biol.222:581-597 (1991) or Griffith etc., EMBO J.12:725-734 (1993) described technology can make up without the V gene library of people's donor of immunity with separate the antibody that obtains at the multiformity array of antigen (comprising autoantigen).Also can be referring to U.S. Patent number 5565332 and 5573905.

[0171] people's antibody also can produce (referring to U.S. Patent number 5567610 and 5229275) by external activating B cell.

Antibody fragment

[0172] develops various technology and prepared antibody fragment.Traditionally the protease digestion by complete antibody derive these fragments (referring to, for example, Morimoto etc., Journal of Biochemical andBiophysical Methods 24:107-117 (1992) and Brennan etc., Science, 229:81 (1985)).Yet the prominent chief cell of the present available reorganization of these fragments is directly produced.For example, antibody fragment can separate from above-mentioned antibody phage library.Perhaps, can directly reclaim Fab '-SH fragment and chemical coupling from escherichia coli and form F (ab ') ₂Fragment (Carter etc., Bio/Technology 10:163-167 (1992)).According to other method, F (ab ') ₂Fragment can directly be separated from the recombinant host cell culture.The other technologies of making antibody fragment will be that the technical staff is conspicuous.In other embodiments, the antibody of selection is strand Fv fragment (scFv).Referring to WO1993/16185 and U.S. Patent number 5571894 and 5587458.Antibody fragment also can be " a linear antibody ", for example, and as described in U.S. Patent number 5641870.This linear antibody fragment can be monospecific or bispecific.

Bi-specific antibody

[0173] bi-specific antibody is the antibody that at least two different epi-positions is had binding specificity.Exemplary bi-specific antibody can be in conjunction with two different epi-positions of T4 antigen.Other these antibody-likes can be in conjunction with CD4 and further combined with second kind of T cell surface marker.Bi-specific antibody also can be used to medicine or cytotoxic agent are positioned the T cell; These antibody have CD4-brachium conjunctivum and medicine or cytotoxic agent brachium conjunctivum.Bi-specific antibody can prepare (F (ab ') for example according to full length antibody or antibody fragment ₂Bi-specific antibody).

[0174] method for preparing bi-specific antibody known in the art.Traditionally, the generation of total length bi-specific antibody is based on two coexpressions that heavy chain immunoglobulin-light chain is right, and wherein two chains have different specificity (Millstein etc., Nature, 305:537-539 (1983)).Because the random assortment of heavy chain immunoglobulin and light chain, these hybridomas (four source hybridomas (quadroma)) have produced the potential mixture of 10 kinds of different antibodies molecules, wherein have only a kind of correct bispecific structure that has.The purification of this kind correct molecule (carrying out with affinity chromatography usually) quite bothers and yields poorly.Similar approach is disclosed in WO 1993/08829 and Traunecker etc., EMBO J., 10:3655-3659 (1991).

[0175], will have the antibody variable region of required binding specificity (antibody-antigen binding site) or merge with the constant region for immunoglobulin sequence according to a kind of diverse ways.Preferably with contain hinge region at least, the constant region for immunoglobulin of the part in CH2 and CH3 district merges.In a kind of method, at least one fusions, exist and contain heavy chain first constant region (CH1) of light chain in conjunction with necessary site.Can with the DNA of coding heavy chain immunoglobulin fusions and, if necessary, the DNA of coding light chain immunoglobulin inserts in the different expression vectors, cotransfection is gone in the suitable hosts biology.When making up three polypeptide chains that usage ratio do not wait so that best yield to be provided, this method provides very big motility for the mutual ratio of regulating these three polypeptide fragments in force.Yet, when at least two polypeptide chains are expressed when producing high yield or this ratio and do not have special meaning with same ratio, the coded sequence of two or all three polypeptide chains can be inserted in the expression vector.

[0176] in an embodiment of this method, is hybrid immunoglobulins heavy chain on this pair characteristic antibody one arm, and is that hybrid immunoglobulins heavy chain-light chain is to (providing second binding specificity) on another arm with first binding specificity.Find, thereby provide easy separation method, required bispecific chemical compound during this dissymmetrical structure helps separating from unwanted immunoglobulin chain mixture because light chain immunoglobulin only is present in half of this pair characteristic molecule.This method is described in WO1994/04690.Produce bi-specific antibody other details can referring to, for example, Suresh etc., Methodsin Enzymology, 121:210 (1986).

[0177] according to the another kind of method of describing in the U.S. Patent number 5731168, the interface between a pair of antibody molecule can be by engineered to improve the heterodimer ratio that reclaims from the reconstitution cell culture as far as possible.A kind of this type of interface comprises antibody constant region C _H3At least a portion of domain.In the method, the one or more little amino acid side chain on the first antibody molecule interface is substituted by larger side chain (for example tyrosine or tryptophan).Substituting big amino acid side chain with less amino acid side chain (for example alanine or threonine) can produce on second antibody molecule interface and this big side chain volume identical or similarly compensatory " hole ".Than other unwanted end-product such as homodimer, can improve the output of heterodimer like this.

[0178] bi-specific antibody comprises crosslinked or " assorted coupling (heteroconjugate) " antibody.For example, a kind of antibody in the assorted conjugate can with the avidin coupling, another kind of antibody can with the biotin coupling.For example, existing people proposes to make the immune system cell targeting be harmful to cell (U.S. Patent number 4676980) with this antibody, and treatment HIV infects (WO 1991/00360, WO 1992/200373 and EP 03089).Assorted coupling antibody can adopt any conventional cross-linking method preparation.Suitable crosslinking agent is well known in the art, and is described in for example U.S. Patent number 4676980, has wherein also described many crosslinking technologicals.

[0179] also describes in the literature from the technology of antibody fragment generation bi-specific antibody.For example, bi-specific antibody can adopt chemistry to connect preparation.Brennan etc., Science, 229:81 (1985) have described Proteolytic enzyme cutting complete antibody to produce F (ab ') ₂Segmental method.When having dithiol chelating agent sodium arsenite, reduce these fragments with near the dithiol stable and prevent to form intermolecular disulphide.Then Fab ' the fragment that produces is changed into sulfo-nitrobenzoyl acid esters (TNB) derivant.Then with mercaptoethylmaine reduction with Fab '-TNB derivant change into Fab '-mercaptan again and again, it is mixed with equimolar other Fab '-TNB derivant with the formation bi-specific antibody.The reagent of the bi-specific antibody useful as selective immobilized enzyme that makes.

[0180] associate reconstitution cell culture directly preparation and the various technology of separating bispecific antibody fragment have been described.For example, bi-specific antibody can adopt the leucine zipper preparation.Kostelny etc., J.Immunol., 148 (5): 1547-1553 (1992).By gene fusion the Fab ' part from Fos and the proteic leucine zipper peptide of Jun and two kinds of different antibodies is linked together.Reduce this antibody homodimer with the formation monomer at hinge region, and then oxidation forms the antibody heterodimer.This method also can be used to prepare the antibody homodimer.Hollinger etc., Proc.Natl.Acad.Sci.USA, " bispecific antibody " technology that 90:6444-6448 (1993) describes is the another kind of method of preparation bispecific antibody fragment.These fragments comprise by too short and can't make that paired joint is connected in light chain variable domain (V between two domains on the same chain _L) weight chain variable domain (V _H).Therefore, the V on fragment _HAnd V _LDomain is forced to and another segmental complementary V _LAnd V _HThe domain pairing, thus two antigen binding sites formed.The additive method that utilizes strand Fv (sFv) dimer to prepare bispecific antibody fragment is also reported.Referring to Gruber etc., J.Immunol., 152:5368 (1994).

[0181] can consider the antibody that two valencys are above.For example, can prepare three-specific antibody.Tutt etc., J.Immunol.147:60 (1991).

The conjugate of antibody and other modifications

[0182] antibody that is used for this method or is included in goods described herein can be chosen wantonly and drug coupling, for example, and as described in WO2004/032828 and U.S. Patent Application Publication No. 2006/0024295.Antibody of the present invention also can with the prodrug activating enzymes coupling that prodrug (for example peptide acyl chemotherapeutics is seen WO 1981/01145) can be changed into the active anticancer medicine.Referring to, for example, WO 1988/07378, U.S. Patent number 4975278, and U.S. Patent Application Publication No. 2006/0024295.

[0183] also can consider other modifications of antagonist here.For example, antibody can be connected in a kind of in the copolymer of various nonprotein polymer such as Polyethylene Glycol (PEG), polypropylene glycol, polyoxyalkylene or Polyethylene Glycol and polypropylene glycol.

[0184] antibody described herein also can be made into liposome.The liposome that contains antibody prepares by means known in the art, as is described in Epstein etc., Proc.Natl.Acad.Sci.USA, 82:3688 (1985); Hwang etc., Proc.Natl.Acad.Sci.USA, 77:4030 (1980); U.S. Patent number 4485045 and 4544545; And the WO 1997/38731 of 1997/10/23 announcement.Liposome with circulation time prolongation is described in U.S. Patent number 5013556.

[0185] by reverse phase evaporation with comprising phosphatidylcholine, the lipid composition of the deutero-PHOSPHATIDYL ETHANOLAMINE of cholesterol and PEG-(PEG-PE) can make useful especially liposome.Extrude liposome has required diameter with generation liposome by the fiber of predetermined hole diameter.As Martin etc., J.Biol.Chem.257:286-288 (1982) is described, can make the Fab ' fragment and the liposome coupling of antibody of the present invention by disulfide exchange reaction.Can choose wantonly in the liposome and comprise chemotherapeutics.Referring to Gabizon etc., J.National Cancer Inst.81 (19) 1484 (1989).

[0186] also considers protein described herein or peptide antibody are carried out amino acid sequence modifications.For example, may need to improve the binding affinity and/or the other biological characteristic of antibody.Introduce in antibody nucleic acid that suitable nucleotide changes or by the synthetic aminoacid sequence variant that can prepare antibody of peptide.This modification comprises, for example, and deletion and/or insert and/or replace residue in the antibody aminoacid sequence.The combination in any that can prepare deletion, insert and replace is to obtain final construction, as long as this final structure has desirable characteristics.Aminoacid changes the translation post-treatment process that also may change antibody, as changing the number or the position of glycosylation site.

[0187] identify that some residue or the regional a kind of process useful as the useful position of mutation is called " alanine scanning mutagenesis " in the antibody, as Cunningham and Wells Science, 244:1081-1085 (1989) is described.At this moment, identify residue or target residue group (for example, charged residue is as arg, asp, his, lys and glu), and with neutrality or negative charge amino acid replacement (most preferably being alanine or poly-alanine) to influence aminoacid and antigenic interaction.Those amino acid positions that show function sensitive for replacement are optimized by further importing other mutant in the replacement site then.Like this, though pre-determined the site that imports the aminoacid sequence sudden change, do not need to pre-determine the character of sudden change itself.For example, carry out the required activity that alanine scans or random mutagenesis also screens the antibody variants of expressing at target codon or zone in order to analyze certain given site mutation performance.

[0188] insertion of aminoacid sequence comprises in the sequence of the fusion (length range from a residue to the polypeptide that comprises 100 or more residues) of amino and/or carboxyl terminal and single or multiple amino acid residues and inserting.The example that inserts of end does not comprise having the antibody that N does not hold the methionyl residue, or the antibody that merges with the cytotoxicity polypeptide.Other insertion variants of antibody molecule comprise with the enzyme of the serum half-life that can increase antibody or the N or the C of polypeptide antibody does not hold fusion.

[0189] another kind of variant is the aminoacid replacement variant.These variants have at least an amino acid residue to be substituted by different residues in antibody molecule.Replace the most interested site of mutation for antibody and comprise hypervariable region, but also consider replacing of FR.The conservative replacement sees Table 1 " conservative replacement " hurdle.If these replacements cause biological activity to change, then can introduce more substantial variation and screening product, these variations are called " exemplary replacement " or as hereinafter divide the apoplexy due to endogenous wind more detailed description at aminoacid in table 1.

[0190] table 1. aminoacid replacement

[0191] substance of antagonist biological characteristics is modified and can be finished by selecting for the effect of keeping following several respects visibly different replacement: the structure that (a) replaces regional polypeptide main chain, for example, be lamella or helical conformation, (b) electric charge of target site molecule or hydrophobicity, or (c) volume of side chain.Can be according to the similarity of amino acid side chain characteristic with them grouping (A.L.Lehninger, Biochemistry (biochemistry), second edition, the 73-75 page or leaf, Worth Publishers (being worth publishing house), New York (1975)): (1) is nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) no charge polarity: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidity: Asp (D), Glu (E); (4) alkalescence: Lys (K), Arg (R), His (H).

[0192] or, can be according to the residue grouping of the characteristic of common side chain: (1) hydrophobicity: nor-leucine, Met, Ala, Val, Leu, Ile with natural generation; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidity: Asp, Glu; (4) alkalescence: His, Lys, Arg; (5) influence the localized residue of chain: Gly, Pro; (6) armaticity: Trp, Tyr, Phe.

[0193] member replacing one of these classifications of non-conservation changes another kind of other member into, and conservative replaces a member with one of these classifications and changes another member in the same classification into.Have that non-conservation or conservative replace, deletion or add (changed, added or deleted in the amino acid residue of any CD4 antibody described herein single amino acids or than the aminoacid (be usually less than 5%, more generally be lower than 4%, 2% or 1%) of small scale) non-expendable CD4 antibody also be applicable to method of the present invention.

[0194] any cysteine residues that does not participate in keeping the correct conformation of antibody also can be substituted, and replaces with serine usually, with the oxidation stability that improves molecule and prevent crosslinked unusually.On the contrary, can in antibody, add the cysteine key to improve its stability (especially when antibody is antibody fragment such as Fv fragment).

[0195] one type replacement variant comprises the one or more hypervariable region residues that replace parental generation antibody.Usually, selecting to be used for the gained variant of further exploitation will have improved biological characteristics with respect to the parental generation antibody that produces them.The method that makes things convenient for that produces such replacement variant is to adopt the affinity maturation of phage display.In brief, can carry out the sudden change in several hypervariable regions site (as the 6-7 site) to produce all possible amino the replacement in each site.So the antibody variants that produces is showed from the filobactivirus granule with the unit price form, is illustrated as the M13 gene III product fusant that is wrapped in each granule.The biological activity of the variant of screening phage display as described herein then (as, binding affinity).In order to identify candidate's hypervariable region site of modifying, can carry out alanine scanning mutagenesis to identify the hypervariable region residue that antigen is combined with obvious contribution.Perhaps, or in addition, analyze the crystal structure of antigen-antibody complex to identify that the contact point between antibody and the antigen may be favourable.According to technology described herein, this contact residues and adjacent residues are the candidates that replaces.In case produce such variant, screening variant group as described herein and in one or more correlation tests, select antibody and further develop with good characteristic.

[0196] amino acid variant of the other types of antibody has changed the original glycosylation pattern of antibody.This change comprises the anti-intravital one or more sugar moieties of deletion, and/or adds non-existent glycosylation site in one or more antibody.

[0197] normally N-connection or O-connection of the glycosylation of polypeptide.N-connects and refers to that sugar moieties is incorporated into the side chain of asparagine residue.Tripeptide sequence agedoite-X-serine and agedoite-X-threonine (wherein X is any aminoacid except that proline) is the bonded recognition sequence of enzymatic of sugar moieties and agedoite side chain.Like this, exist one of these tripeptide sequences to produce potential glycosylation site in the polypeptide.O-connects glycosylation and is meant that a kind of sugar in N-acetylgalactosamine, galactose or the xylose is incorporated into hydroxy-amino-acid, and modal is serine or threonine, though also available 5-hydroxyproline or 5-hydroxylysine.

[0198] can make it comprise one or more above-mentioned tripeptide sequences to be not difficult to be implemented in and to add glycosylation site (connecting glycosylation site) in the antibody by changing aminoacid sequence usually for N-.Also can in the original antibody sequence, add one or more serines or threonine residues or replace and carry out this change (connecting glycosylation site) for O-with one or more serines or threonine residues.

[0199] when antibody comprised the Fc district, the sugar that it connected can be changed or remove.For example, in a kind of glycosylation variant as herein described, one or more aminoacid replacement in the Fc district of antibody, have been introduced to remove one or more glycosylation sites.The effector function of this nonglycosylated antibody reduces, for example compare, thereby it induces Cytotoxic ability of complement activation and/or antibody dependent cellular mediation to reduce with the human IgG1, and nonglycosylated antibody and Fc receptor combine reduction (or not having).

[0200], for example,, may need it is modified to strengthen the ADCC and/or the CDC of antibody in the methods of the invention as the expendable antibody of second chemical compound for some antibody.For example, U.S.

2003/0157108 (Presta L.) has described the antibody with the ripe carbohydrate structure that lacks the fucose be incorporated into the antibody Fc district.Also can referring to U.S.2004/0093621 (Kyowa Hakko Kogyo Co., Ltd.).Mentioned the antibody that in the sugar that is incorporated into the antibody Fc district, has five equilibrium N-acetylglucosamine (GlcNAc) in the U.S. Patent number 6602684 of the WO2003/011878 of Jean-Mairet etc. and Umana etc.The antibody that has at least one galactose residue in the oligosaccharide that is incorporated into the antibody Fc district is reported in the WO1997/30087 of Patel etc., also can be referring to WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.), they relate to the antibody that the Fc district is combined with the sugar of change.

[0201] therefore, the glycosylation variant is optional to comprise the Fc district, and the sugar that wherein is incorporated into the Fc district is in conjunction with lacking fucose.This variant has improved ADCC function.Randomly, the Fc district also comprises one or more aminoacid replacement with further improvement ADCC, and for example, the 298th, 333 and/or 334 in the Fc district (the Eu numberings of residue) replace.The example that relates to the publication of " taking off fucosylated " or " fucose defective " antibody comprises: U.S.2003/0157108; WO 2000/61739; WO 2001/29246; U.S.2003/0115614; U.S.2002/0164328; U.S.2004/0093621; U.S.2004/0132140; U.S.2004/0110704; U.S.2004/0110282; U.S.2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; Okazaki etc., J.Mol.Biol.336:1239-1249 (2004); With Yamane-Ohnuki etc., Biotech.Bioeng.87:614 (2004).The example for preparing the cell line of taking off fucosylated antibody comprises Lec13 Chinese hamster ovary celI (Ripka etc., the Arch.Biochem.Biophys.249:533-545 (1986) of the fucosylated defective of protein; U.S.2003/0157108, Presta, L; With WO 2004/056312, Adams etc., especially embodiment 11), and knock out cell line, as α-1,6-fucosyl transferase gene FUT8-knocks out Chinese hamster ovary celI (Yamane-Ohnuki etc., Biotech.Bioeng.87:614 (2004)).

[0202] modification of antagonist effector function for example for strengthening the ADCC and/or the CDC of antibody, can realize by introduce one or more aminoacid replacement in the antibody Fc district.Perhaps, or in addition, cysteine residues can be introduced into the Fc district, thereby forms interchain disulfide bond in this zone.Cell killing and ADCC that the same dimerization antibody that generates like this has the complement-mediated of improved internalization ability and/or raising.Referring to Caron etc., J.Exp Med.176:1191-1195 (1992) and Shopes, B.J.Immunol 148:2918-2922 (1992).Same dimerization antibody with enhanced anti-tumor activity also can be according to Wolff etc., and the description of Cancer Research53:2560-2565 (1993) adopts the Heterobifunctional cross linker to prepare.Perhaps, thus antibody engineering can be built into and have dual Fc district and have dissolving of enhanced complement and ADCC ability.Referring to Stevenson etc., Anti-Cancer Drug Design 3:219-230 (1989).(Presta L.) has described the antibody of the ADCC function that has improvement when having people effector lymphocyte to WO 2000/42072, and this antibody comprises aminoacid replacement in its Fc district.Preferably, the antibody with ADCC of improvement comprises replacement 298,333 and/or 334 of Fc district.Preferably, the Fc district of described change comprises the human IgG1 Fc district that replaces or be made of described replacement in these positions 1,2 or 3.

[0203] has the Clq combination of change and/or the antibody of CDC and be described in WO 1999/51642 and U.S. Patent number 6194551,6242195,6528624 and 6538124 (Idusogie etc.).Comprise aminoacid replacement on this antibody one or more in its Fc district amino acid position 270,322,326,327,329,313,333 and/or 334.The non-expendable anti-CD 4 antibodies that comprises this aminoacid replacement has constituted an embodiment of the invention.

[0204], can mix antibody (especially antibody fragment) as remedying the receptors bind epi-position as described in the U.S. Patent number 5739277 for example for prolonging the serum half-life of antibody.In the literary composition, term " is remedied the receptors bind epi-position " and is referred to be responsible for prolonging the epi-position that IgG divides IgG molecule (for example, IgG1, IgG2, IgG3 or IgG4) the Fc district of serum half-life in the daughter.Have in its Fc district the antibody that replaces and have the serum half-life of prolongation also be described in WO2000/42072 (Presta, L.).Comprise this non-expendable anti-CD 4 antibodies of remedying the receptors bind epi-position and constituted an embodiment of the invention.

[0205] any non-expendable (or other) of the present invention antibody in the Fc district, can comprise at least one replace with improve FcRn in conjunction with or serum half-life, for example, non-expendable anti--CD4 variant antibody.For example, the present invention also provides the antibody that comprises variant Fc district that neonatal Fc receptor (FcRn) binding affinity changes.The similar of FcRn constitutes in major histocompatibility complex (MHC) and by α-chain and the non-covalent combination of B2M.Ghetie and Ward (2000) Annu.Rev.Immunol.18:39-766 are seen in the summary of the multiple function of neonatal Fc receptor FcRn.Passive child's FcRn of flowing to and regulating the serum IgG level plays a role from mother at immunoglobulin IgG.FcRn is as a kind of receptor of remedying, in conjunction with and transport in the cell and stride cell by the complete IgG of endocytosis, and from acquiescence degradation pathway rescue IgG.Although be responsible for remedying the machine-processed not clear of IgG, think unconjugated IgG in lysosome by Proteolytic enzyme, and bonded IgG is recycled to cell surface and discharges.This control action occurs in the middle of the endotheliocyte that spreads all over adult's tissue.FcRn expresses in liver, mammary gland and adult's intestinal at least.FcRn is in conjunction with IgG; As if FcRn-IgG interacts and fully studies, relate to the residue on IgG Fc district CH2, the CH3 domain interface.These residues interact with the residue that mainly is arranged in FcRn α 2 domains.

[0206] In some embodiments of the present invention, non-expendable anti--CD4 variant antibody show with FcRn combine enhancing and in Fc district amino acid position 238,256,265,272,286,303,305,307,311,312,317,340,356,360,362,376,378,380,382,413,424 or 434 any one or a plurality of on comprise amino acid modified, wherein, the numbering of Fc district residue is the EU index number as the Kabat numbering.Referring to, for example, United States Patent (USP) 6737056; With Shields etc., J.Biol.Chem.276:6591-6604 (2001).In an embodiment of the invention, antibody comprises variant IgGFc district, wherein comprises the aminoacid replacement (N434H) of Asn 434 → His at least.In an embodiment of the invention, antibody comprises variant IgG Fc district, wherein comprises the aminoacid replacement (N434A) of Asn 434 → Ala at least.Usually, these variants are higher than the antibody in the Fc district with native sequences/wild-type sequence to the binding affinity of FcRn.The advantage of these Fc variant polypeptides and antibody is and can be remedied and recirculation, rather than is degraded.These non-expendable resisting-CD4 variant antibody can be used for method provided herein.An example of non-expendable CD4 variant antibody is that any TRX1 antibody as herein described can comprise replacement on heavy chain position 434, as N434A or N434H.

[0207] the serum albumin binding peptide is mixed the serum half-life that antibody also can improve antibody, as U. S. application serial number 20040001827 (Dennis, M.) described.The non-expendable anti-CD 4 antibodies that contains this serum albumin binding peptide has constituted an embodiment of the invention.

[0208] also considered to have the engineered antibody (US2002/0004587 A1, Miller etc.) of 3 or a plurality of (preferred 4) functional antigen binding site.The non-expendable anti-CD 4 antibodies that comprises this a plurality of antigen binding sites has constituted an embodiment of the invention.

[0209] prepared the nucleic acid molecules of encoding antibody aminoacid sequence variant by various means known in the art.These methods include, but not limited to prepare from natural origin separation (for the aminoacid sequence variant of natural generation) or by the antibody variants of preparation earlier or oligonucleotide mediated (or fixed point) mutation, PCR mutation and the cassette mutagenesis of non-variant form.

[0210] puts into practice when of the present invention, can choose the many routine techniquess that adopt in molecular biology, microbiology and the recombinant DNA technology wantonly.These technology be know and be set forth in, for example, Berger and Kimmel, Guide to Molecular Cloning Techniques (molecule clone technology guide), Methods inEnzymology (Enzymology method), the 152nd volume, the academic press, Santiago, California; Sambrook etc., Molecular Cloning-ALaboratory Manual (molecular cloning-laboratory manual) (third edition), the 1-3 volume, cold spring harbor laboratory (Cold Spring Harbor Laboratory), the cold spring port, New York, 2000 and the Current Protocols in Molecular Biology (newly organized molecular biology experiment guide) that compiles such as F.M.Ausubel, CurrentProtocols (newly organized experiment guide series), (GreenePublishing Associates is Inc.) with (the John Wiley of John Willie father and son company in Green publishing company; Sons, Inc.) combined publication, (supplementary issue after 2006).About for example cell separation and cultivation (for example, be used for follow-up nucleic acid or Separation of Proteins) other useful reference materials comprise Freshney (1994) Culture of AnimalCells (cultivation of zooblast), Manual of Basic Technique (basic technology handbook), the third edition, Willie Li Si company (Wiley-Liss), New York, and the reference material of wherein quoting; Payne etc., (1992) Plant Cells and Tissue Culture in Liquid Systems (cultivating plant cell and tissue), John Willie father and son company, New York, New York at liquid system; Gamborg and Phillips (volume) (1995) Plant Cell, Tissue and Organ Culture (cultivation of plant cell, tissue and organ); Fundamental MethodsSpringer Lab Manual (Springer laboratory manual basic skills), Springer-Verlag (Berlin, the Heidelberg, New York) and Atlas and Parks (volume) The Handbook of Microbiological Media (microorganism culturing handbook) (1993) CRC publishing house (CRC Press), rich card Leiden (Boca Raton), the Florida State.The method for preparing nucleic acid (for example, by amplification in vitro, from cell purification or chemosynthesis), the method (for example, direct mutagenesis, Restriction Enzyme digestion, connection etc.) of operation nucleic acid and the various carriers, cell line etc. that are used for operating and preparing nucleic acid are described in above-mentioned list of references.In addition, can or buy any basically polynucleotide (comprise, for example, labelling or biotinylated polynucleotide) from the customization of any commercial source.

Administration

[0211] one of ordinary skill in the art will be understood, and the suitable dose of non-expendable CD4 antibody roughly is already used dosage in the clinical treatment usually, and these clinical treatments give similar antibody separately or with combination with other therapeutic agents.Has various dose according to the disease that will treat.The doctor who treats can determine to be used for the suitable dose of each object.The preparation of commercially available second chemical compound that gives with described non-expendable CD4 antibody combination and dosage can be as rule of thumb determining as described in the description of manufacturer or by experienced doctor.

[0212] be prevention or treatment disease, the purpose that the suitable dose of second chemical compound that the combination of antibody and any and non-expendable antibody gives will depend on the aforesaid disease type that will treat, severity of disease and process, give non-expendable antibody or combination is to prevent or treatment, former treatment, patient's clinical history and the reaction of antagonist or compositions and the doctor in charge's decision.Non-expendable antibody or combination are fit to disposable or are more typically in a series of treatments give the patient.

[0213] according to the type and the seriousness of disease, the initial candidate's dosage that gives patient's non-expendable CD4 antibody is about 1 μ g/kg to 50mg/kg (for example 0.1-20mg/kg), can give by the dispenser that one or many separates, perhaps can give by continuous infusion.Typical case's daily dose may be that about 1 μ g/kg arrives about 100mg/kg or higher, and this depends on above-mentioned factor.For at a couple of days or longer time (depending on disease) repeat administration, sustainable treatment obtains the inhibition that needs up to disease symptoms.Yet other dosages also are useful.Usually, the clinician gives antibody of the present invention (separately or with second chemical compound combination) and reaches up to its dosage required biological effect can be provided.Can detect the progress of therapy of the present invention easily by routine techniques and test.

[0214] for example, the description that can choose wantonly according to U.S. Patent Application Publication No. 2003/0108518 or 2003/0219403 of the non-expendable CD4 antibody of TRX1 gives.In one embodiment, give object with the combination of second chemical compound separately or as mentioned above with 3-5mg/kg (every kilogram of object body weight administered antibodies milligram number), continued treatment is up to the inhibition that required disease symptoms occurs.Described non-expendable antibody can be chosen wantonly and give a period of time to keep antibody horizontal suitable in the object (perhaps, if antibody is used in combination with second chemical compound, then be the level of the suitable antibody and second chemical compound combination) thus realize or keep inhibition to symptom.

[0215] described non-expendable CD4 antibody can give by any suitable pathways, comprises in parenteral, part, subcutaneous, intraperitoneal, the lung, intranasal and/or intralesional administration.The parenteral infusion comprises intramuscular, intravenous, intra-arterial, intraperitoneal or subcutaneous administration.Also can adopt intrathecal drug delivery (referring to, for example, the U.S. Patent Application Publication No. 2002/0009444 of Grillo-Lopez).In addition, this antibody also is fit to by the administration of pulse infusion, for example to reduce the mode of antibody dosage gradually.Preferably, intravenous or subcutaneous administration preferably pass through intravenous infusion administration.Each medication can be adopted identical or different medication.In one embodiment, each medication gives by intravenous.

[0216] as mentioned above, non-expendable CD4 antibody can give separately or give with at least a second chemical compound combination.The dosage of these second chemical compounds and route of administration usually with use before identical, or be about 1-99% of using dosage before.If use this second chemical compound, preferably its consumption is lower than the consumption when not containing non-expendable CD4 antibody, so that eliminate or reduce the side effect that causes thus.

[0217] still as mentioned above, many second suitable chemical compounds are known in the art, and the dosage of this second chemical compound and medication were equally described.An example is to give non-expendable CD4 antibody with treatment lupus (or MS, rheumatoid arthritis or inflammatory bowel or above-mentioned other diseases) with the cyclophosphamide combination.Many cyclophosphamide therapeutic schemes had been described in the document.Exemplary scheme includes, but not limited to continue 6 months every month one time intravenous and gives 0.5-1.0g/m ², then per 3 months of 30 middle of the month once; With continue 3 months whenever biweekly intravenous give 500mg; Continued for 12 weeks or 6 months in orally give 1-3mg/kg/ days.Referring to, for example, Petri (2004) " Cyclosphosphamide:newapproaches for systemic lupus erythematosus (cyclophosphamide: the new method of therapy system lupus erythematosus) " Lupus 13:366-371 and Petri and Brodsky (2006) " High-dosecyclophosphamide and stem cell transplantation for refractory systemic lupuserythematosus (the intractable systemic lupus erythematosus (sle) of high dose cyclophosphamide and cellular replacement therapy) " JAMA 295:559-560.

[0218] non-expendable anti-CD 4 antibodies and any second chemical compound of the present invention can adopt identical or different route of administration to give simultaneously, for example as single compositions or as two or more different compositionss.Perhaps, or in addition, can any order successive administration.In some embodiment, the blanking time that gives between two or more compositionss can be from several minutes to a couple of days, several weeks or several months.For example, non-expendable anti-CD 4 antibodies can at first give, and gives second chemical compound of the present invention then.Yet, also can give or at first give second chemical compound of the present invention simultaneously.

[0219] as mentioned above, can choose wantonly with the combination of non-expendable CD4 antibody and second chemical compound give the third, the 4th kind of chemical compound, or the like.Similarly, for example during treating with non-expendable CD4 antibody or combination, can treat lupus (for example, spasm, incontinence, pain, fatigue) or the Secondary Symptom or the related symptoms of MS, rheumatoid arthritis, inflammatory bowel or other symptoms or disease of object.

Pharmaceutical preparation

[0220] will have the non-expendable CD4 antibody of required purity and pharmaceutically acceptable carrier, excipient or the stabilizing agent (Remington ' s Pharmaceutical Sciences (Lei Mingdun pharmaceutical science) that chooses wantonly, the 16th edition, Osol, A. compiles (1980)) thus mix and the therapeutic agent that is used for antibody of the present invention can be made lyophilized formulations or the aqueous solution form that is used to store.Acceptable carrier, excipient or stabilizing agent are nontoxic to the receptor under used dosage and concentration, comprising: buffer agent, and as phosphoric acid (salt), citric acid (salt) and other organic acid; Antioxidant comprises ascorbic acid and methionine; Antiseptic is (as octadecyl dimethyl benzene ammonio methacrylate; Chloor-hexaviet; Benzalkonium chloride, benzethonium chloride; Phenol, butanols or benzylalcohol; Alkyl paraben such as methyl parahydroxybenzoate or propyl p-hydroxybenzoate; Catechol; Resorcinol; Hexalin; The 3-amylalcohol; With-cresol); Low-molecular-weight (being less than about 10 residues) polypeptide; Protein, as serum albumin, gelatin, or immunoglobulin; Hydrophilic polymer, the pyrroles burns ketone as polyethylene; Aminoacid, as glycine, glutamine, agedoite, arginine, or lysine; Monosaccharide, disaccharide and other carbohydrate comprise glucose, mannose, or dextrin; Chelating agen is as EDTA; Saccharide is as sucrose, mannitol, trehalose or Sorbitol; Salify counter ion counterionsl gegenions such as sodium; Metal complex (for example Zn-protein complex); And/or non-ionic surface active agent, as Tween ^TM,

Or PEG.

[0221] be fit to the subcutaneous lyophilized formulations that gives and be described in, for example, U.S. Patent number 6267958 (Andya etc.).This lyophilized formulations can be reconstructed into increased protein concentration with suitable diluent, gives the mammal that will treat with the preparation of rebuilding is subcutaneous then.Also considered the crystal form of antibody.Referring to, for example, U.S.2002/0136719A1 (Shenoy etc.).

[0222] preparation described herein also can contain to some extent essential at least a second chemical compound of specific adaptations disease of treatment, preferably have complementary activity and each other can otherwise impact those.For example, may also need to provide cytotoxic agent (for example methotrexate, cyclophosphamide or azathioprine), chemotherapeutics, immunosuppressant, cytokine, cytokine antagonist or antibody, somatomedin, hormone, integrin, integrin antagonist or antibody are (for example, LFA-1 antibody, or alpha-4 integrin antibody such as natalizumab), interferons medicine such as IFN-β-1a or IFN-β-1b, oligopeptide such as acetic acid glatiramer, intravenous immunoglobulin (gamma Globulin), lymphocyte-expendable medicine (for example, mitoxantrone, cyclophosphamide

Antibody, or carat Qu Bin), non-lymphocyte-the expendable immunosuppressant is (for example, MMF or ciclosporin), the cholesterol of " his spit of fland " class reduces medicine, estradiol, the treatment lupus, MS, rheumatoid arthritis or inflammatory bowel Secondary Symptom or related symptoms are (for example, spasm, incontinence, pain, fatigue) medicine, tnf inhibitor, DMARD, NSAID, 17-hydroxy-11-dehydrocorticosterone (for example, methylprednisolone, prednisone, dexamethasone, or glucocorticoid), levothyrocine, cyclosporin A, rainbow trout somatostatin (somatastatin) analog, anti--metabolite, T-or B-cell surface antagonist/antibody etc., or above-mentioned at other medicaments described in preparation one joint.The type and the effective dose of this other medicaments depend on, for example, and the type of the antibody amount that exists in the preparation, the lupus that will treat or MS or other diseases or disease and the clinical parameter of object.

[0223] active component can be wrapped up for example by in condensation technique or the microcapsule by the interfacial polymerization preparation, respectively for example, the colloid drug delivery system (for example, liposome, albumin microsphere, microemulsion, nano-particle and Nano capsule) or thick Emulsion (macroemulsion) in hydroxy methocel or gelatin-microcapsule and poly--(methylmethacrylate) microcapsule.This technical description in, for example, Remington ' sPharmaceutical Sciences (Lei Mingdun pharmaceutical science), the 16th edition, Osol, A. compiles (1980).

[0224] can prepare slow releasing preparation.The suitable example of slow releasing preparation comprises the semi-transparent substrate of the solid hydrophobic polymer that contains non-expendable antibody, and the form of described substrate is a formed article, as film or microcapsule.The example of sustained-release matrix (for example comprises polyester, hydrogel, poly-(2-ethoxy-methacrylate), or poly-(vinyl alcohol)), polyactide (U.S. Patent number 3773919), the copolymer of L-glutamic acid and y ethyl-L-glutamic acid, nondegradable ethane-acetic acid ethyenyl ester, degradable lactic acid-hydroxyacetic acid copolymer such as Lupron

(Injectable microspheres that constitutes by lactic acid-hydroxyacetic acid copolymer and leuprorelin acetate), and poly--D-(-)-3-hydroxybutyric acid.

[0225] preparation that is used for vivo medicine-feeding must be aseptic.This can realize easily by aseptic membrane filtration.

Manufacture

[0226] in other embodiments of the present invention, provides the manufacture that contains the material that is used for the treatment of lupus, MS, rheumatoid arthritis, inflammatory bowel or above-mentioned other diseases or disease.Preferably, this manufacture comprises (a) container, and the compositions that comprises non-expendable CD4 antibody and pharmaceutically acceptable carrier or diluent is housed in the container; (b) about by giving the package insert of the operation instruction of lupus, MS, rheumatoid arthritis, inflammatory bowel or other diseases or disease that antibody comes treatment target separately or with at least a second chemical compound combination.

[0227] described package insert is on described container or be attached thereto.Suitable containers comprises, for example, and medicine bottle, bottle, syringe etc.Container can be made as glass or plastics by various materials.Container is equipped with or contains the compositions that can effectively treat lupus, MS, rheumatoid arthritis, inflammatory bowel or other diseases or disease, and has an aseptic access port (for example, described container can be intravenous solution bag or the bottle with stopper that available hypodermic needle pierces through).At least a activating agent of compositions is non-expendable antibody.Label or package insert show that said composition can be used to treat lupus, MS, rheumatoid arthritis, inflammatory bowel or other diseases or the disease for the object of the suitable lattice of treatment, also provides dosage and special instruction at interval about antibody and any other medicine.

[0228] described manufacture also can comprise second container, and pharmaceutically acceptable dilution buffer agent wherein is housed, as water for injection,bacteriostatic (BWFI), phosphate-buffered saline, Ringer's solution and glucose solution.Described manufacture is optional to comprise the second or the 3rd container, and second chemical compound wherein is housed, and as described herein is any, the operation instruction of this manufacture useful second compounds for treating object on package insert.Perhaps, the compositions that comprises non-expendable CD4 antibody also comprises second chemical compound.Described manufacture also can comprise from the other materials of commercial and user viewpoint of measures needs, as comprises other buffers, diluent, filter, syringe needle and syringe.

Embodiment

[0229] should understand, embodiment described herein and embodiment are only for exemplifying purpose, Given this those skilled in the art can obtain prompting and make various improvement or variation, and these improvement or variation are included within the scope of the spirit and scope of this description and accessory claim book.Therefore, following examples only are used for setting forth and unrestricted the present invention.

Embodiment 1: with non-expendable CD4 Antybody therapy lupus independent or combination

[0230] a series of experiments have been listed below to confirm that non-expendable CD4 antibody is effective in the SLE preclinical models.The performance of this antibody is compared with experimental treatment standard substance with exemplary nursing.

[0231] NZBxW F1 mice demonstrates spontaneous lupoid acne nephropathy, efficacy models before providing useful SLE clinical (referring to, for example, Theofilopoulos (1992) " Murine models of systemic lupuserythematosus (the Muridae model of systemic lupus erythematosus (sle)) ", be selected from Systemic LupusErythematosus (systemic lupus erythematosus (sle)), Lahita (volume) Churchill Livingstone, New York, 121-194).Fig. 5 is the sketch map that disease was made progress with the age in this model.Observed symptom comprises that ds-DNA antibody, albuminuria, nephridial tissue pathology, blood urea nitrogen (BUN) rising and mortality rate occurring raises.Arrow represents to begin in two researchs of relatively this antibody and other treatment the time point with non-expendable CD4 Antybody therapy.

[0232] in this model, with effect before the non-expendable CD4 antibody of rat YTS177 clinical (Cobbold etc., (1990) " The induction of skingraft tolerance in MHC-mismatched orprimed recipients:primed T-cells can be tolerized in the periphery with CD4 andCD8 antibodies (induce skin transplantation patience in MHC-mispairing or sensitization receptor: sensitized T cell can be by CD4 and the tolerance of CD8 antibody in the joint slightly at the end) " Eur J Immunol 20:2747-2755) and non-binding control antibodies (contrast Ab or contrast Ig), CTLA4-Ig (in the clinical development) and cyclophosphamide (

, CTX; Present nursing for treating standard substance) compare.The non-expendable CD4 antibody of YTS177 is so kind as to give by the HermanWaldmann of Oxford.Control antibodies is that uncorrelated mice IgG1 antibody (because uncorrelated rat anti is known from experience the immunoreation that causes at himself, influences lysis, therefore adopts mouse antibodies in contrast; Rat anti-CD4 antibody capable prevents this reaction at self).The CTLA4-Ig construction that adopts comprises hinge-C3 with the human IgG1, the ectodomain of the Muridae CTLA-4 that C4 Ig domain merges, and afterwards by Linsley etc., (1991) J Exp Med 174 (3): 561 modelings.

[0233] at 8 months when big, the albuminuria of screening NZB x NZW mice is divided into 5 groups at random according to their albuminuria scoring.That the disease of this moment is considered to is medium-and serious.During the experiment beginning, the urine protein CONCENTRATION DISTRIBUTION of every group of 19 mices is as follows: 32% is〉300mg/dl; 24% is 100-300mg/dl; With 44% be 30-100mg/dl.Mice with control antibodies (contrast Ab or contrast Ig), YTS177 (non-expendable anti--CD4), the combination of CTLA4-Ig, cyclophosphamide (CTX) or anti--CD4 and CTX treatment 6 months continuously.YTS177 and CTLA4-Ig send weekly three times by intraperitoneal (IP) injection with 5mg/kg; Per 10 days of cyclophosphamide (CTX) gives (independent or with shown in the YTS177 combination of dosage) with 50mg/kg IP.The variation of urine protein concentration (for example, albuminuria), blood urea nitrogen (BUN) and the survival rate of monitoring mice.

[0234] as shown in Figure 6, giving non-expendable CD4 antibody has postponed progress time (Fig. 6 A), has improved survival rate (Fig. 6 B), reduced albuminuria (having shown the data for the treatment of back 5th month, Fig. 6 C) and has reduced average BUN (Fig. 6 D).

[0235] can reverse the seriousness lupus nephritis with non-expendable CD4 Antybody therapy, as shown in Figure 7.Fig. 7 A has shown that moment albuminuria shown in the treatment back is lower than the percentage ratio of the mice of 300mg/dl.Separately or give non-expendable CD4 antibody with the cyclophosphamide combination and cause albuminuria the mice of 300mg/dl reduces only, show that the nephritis symptom in the utmost point terminal illness obtains reversing, this does not observe in single group with control antibodies, CTLA4-Ig or cyclophosphamide treatment.Fig. 7 B has shown the percentage ratio for the treatment of in first month the mice of reversing from the 300mg/dl albuminuria.(Fig. 7 B data presented is consistent with four researchs, comprising a research described herein and three similar researchs.Data only comprise when beginning treatment albuminuria〉mice of 300mg/dl.) when CD4 antibody and cyclophosphamide (CTX) combination, observe and reverse albuminuretic cooperative effect.

[0236] combined therapy with non-expendable CD4 antibody and cyclophosphamide also can effectively reduce albuminuria.Fig. 9 has shown treatment albuminuretic multiple comparisons analysis 6 months the time, analyze and adopt the Dunnett method, among Fig. 9 A with cyclophosphamide treatment group as the reference matched group, adopt non-expendable CD4 Antybody therapy group as the reference matched group among Fig. 9 B.The reference contrast is represented with black matrix, only points out to compare the p value that the reference contrast has each group of statistical significance in the drawings.The result reconfirms, non-expendable CD4 antibody is better than CTLA4-Ig (referring to, for example, Fig. 9 B) aspect the albuminuria reducing.This result also proves, the combination of non-expendable CD4 antibody and cyclophosphamide obviously is better than single with cyclophosphamide (referring to, for example, Fig. 9 A) for the albuminuria that reduces model.

[0237] detect kidney segment CD4 and CD8 coloration result and show, with the control antibodies treatment after 4 months between mice renal medulla or pelvis matter lymphocytic infiltration appears.On the other hand, can reduce when treating back 4 months between kidney observed CD4+ cell in the matter with CD4 antibody or with CTLA4-Ig treatment.The CD4 Antybody therapy can not influence the number of observed CD8+T cell in kidney.

[0238] the NZBxWF1 mice (SLE mouse model) that demonstrates spontaneous lupoid acne nephropathy with non-expendable CD4 Antybody therapy also can be limited the ds-DNA antibody titer and raise.As shown in Figure 8, compare with the animal for the treatment of with control antibodies, lower with ds-DNA antibody titer rising in time in the animal of non-expendable CD4 Antybody therapy.Fig. 8 A (having shown the titre (the average log value of each treatment group is approximately 3) when raising) and Fig. 8 B (having shown the titre (the log value of non-expendable resisting-CD4 and control antibodies treatment group is about 3.5 and 4.5 respectively) when treating back 3 months) are compared.In this experiment, treatment is since 6 months but not 8 months when big.

[0239] in addition, reduced the number of activation CD4+T cell in the spleen with the CD4 Antybody therapy, cell number utilizes the proteic antibody of anti-T cell activation relevant surfaces to measure by flow cytometry.As shown in Figure 8, compare with the animal of control antibodies treatment, 3 week of treatment the back in the spleen of the animal of non-expendable CD4 Antybody therapy, find the number of CD4+CD69+ cell (Fig. 8 C) and CD4+CD25+ cell (Fig. 8 D) less (from 8 months time begin treatment) greatly.

[0240] non-expendable CD4 antibody is also effective for the slight disease of treatment except that medium-serious disease.Substantially as mentioned above, with contrast Ab, YTS177 (non-expendable anti--CD4), CTLA4-Ig or cyclophosphamide ( ) to handle 6 months big, albuminuria all be the NZB x NZW mice of 30-100mg/dl.The variation and the survival rate of monitoring murine protein urine.As shown in Figure 6, compared with the control, gave non-expendable CD4 since 6 months when big and can postpone the progress time (Fig. 6 E) and improve survival rate (Fig. 6 F), prove that to give slight disease height with non-expendable CD4 antibody effective.(compared with the control, all treatments are all very effective: in the time of 7 months, and * p＜0.025 of progression of disease time (Fig. 6 E), * p＜0.04 of survival rate (Fig. 6 F).)

[0241] in a word, early stage or late period is all effective for NZB x WF1 mice with non-expendable CD4 Antybody therapy in disease.Can prolong no disease progression (disease-free progression) and survival rate with described Antybody therapy, postpone rising and the brightic generation of BUN, limit the rising of anti--dsDNA titre, and reduce activatory CD4+T cell number.Reducing aspect the albuminuria, observed effect is better than CTLA4-Ig with suitable with the cyclophosphamide treatment during with this Antybody therapy 5 or 6 months; For terminal illness, the difference between anti--CD4 and the CTLA4-Ig is more remarkable.In addition, in the NZB/WF1 of SLE model, non-expendable CD4 antibody and cyclophosphamide combination obviously are better than singly using cyclophosphamide.

Experiment flow

Urinalysis

[0242] with Indiana, USA Ai Er Elkhart city Bayer AG

(IN USA) measures albuminuria to 50 type urochemistry analysers for Bayer Corporation, Elkhart.With the urine of a fresh collection drop in reagent paper (

10SG, Baeyer) on, go immediately reagent paper to be inserted analyser after the unnecessary urine with the suction of clean gauze sponge.

Measure the blood urea nitrogen level

[0243] adopts Cobas

400 type chemical analyzers (Luo Shi diagnostic reagent company (RocheDiagnostics), Basel, Switzerland) and urine examination test agent (also being provided by Luo Shi diagnostic reagent company) are measured blood urea nitrogen according to the explanation of manufacturer.Precinorm ^TMAnd Precipath ^TMLyophilizing human serum contrast (Luo Shi diagnostic reagent company) is used separately as normal and unusual contrast.

The CD4 of kidney segment and CD8 dyeing

When [0244] carrying out CD4/CD8 double labeling immunohistochemistry, downcut the freezing kidney segment of 5 micron thickness, with ice-cooled (20 ℃) acetone fixed 5 minutes, with twice of TBS/0.1% Tween 20 (TBST) rinsing, each 5 minutes, handle 1 hour to seal endogenous oxidase active at 37 ℃ with glucoseoxidase then.The section then with the TBST rinsing and use available from Vector Labs (carrier laboratory (Vector Labs), Bai Lingaimu (Burlingame), California)) avidin/biotin closed reagent box seal endogenous avidin/biotin.After the TBST rinsing, handle 30 minutes down to seal endogenous immunoglobulin in room temperature (RT) with 10% rabbit anteserum/3% BSA/TBS.

During [0245] with the CD8 labelling, to cut into slices and 8ug/ml biotinylation rat anti-mice CD8 monoclonal antibody (MAb), clone 53.6-7 (Fa Ma King Company (Pharmingen), San Diego city (San Diego), California) room temperature together cultivated 1 hour.Negative control adopts natural isotype rat IgG2a as first antiserum.Cultivated 30 minutes with Vectastain ABC-Elite reagent (carrier laboratory) room temperature with cutting into slices after the TBST rinsing.(painted section is observed by Illinois Rockford Pierre Si biotech company (Pierce Biotechnology, Rockford, IL)) as chromogen to use metal enhancement mode DAB then.

When [0246] making the secondary labelling, seal the avidin/biotin (first reaction in) of section once more with the breadboard avidin/biotin closed reagent of carrier box with CD4 antibody.To cut into slices then and 0.5ug/ml rat anti-mice CD4MAb, clone RM4-4 (Fa Ma King Company) room temperature together cultivated 1 hour.Negative control adopts natural isotype rat IgG2b as first antiserum.To cut into slices and TSA with after the TBST rinsing ^TMSucc-PEG-DSPE-HRP complex in (cheese amide amplification of signal (tyramide signal amplification)) test kit (PEL company (Perkin-Elmer LAS Inc.), Boston, Massachusetts) room temperature was together cultivated 30 minutes.After the TBST rinsing, will cut into slices then and biotinylation TSA ^TMAmplifing reagent (PEL company) room temperature was together cultivated 3 minutes, at room temperature carried out second with Succ-PEG-DSPE-HRP then and took turns cultivation in 30 minutes.Use then

Red (carrier laboratory) observes painted section as chromogen.

[0247] with the Myer hematoxylin section of double labeling was redyed 1 minute slightly then, with the tap water rinsing and cover Crystal/Mount (Foster city, California Bai Maide company (Biomeda Corporation, FosterCity, CA)) coverslip.

Measure two strands-dna antibody titre

[0248] measures anti-ds-DNA antibody titer by ELISA.Immunity dull and stereotyped (numbering: in Nunc MAXIsorb 384 holes 464718) with poly-L-Lysine (25 μ l/ holes, 0.01%, Sigma P4707) the room temperature bag was by 1 hour, deionized water wash, dry 1 hour of air at room temperature, use calf thymus DNA (PBS is made into 2.5 μ g/ml for Sigma D1501,25 μ l/ holes) to be spent the night then in 4 ℃ of bags.Pour out the calf thymus DNA solution on the flat board, add 50 μ l sealing buffer (PBS, 0.5% BSA pH7.2), then flat board was vibrated 1 hour in room temperature.Flat board is used lavation buffer solution (PBS, 0.05% Tween then ^TM20 (polyoxyethylene (20) sorbitol anhydride monolaurates), pH7.2) washing is three times.

[0249] with assay buffer (PBS, 0.5% BSA, 0.05% Tween ^TM20,0.01% Procline3000) serial dilution of preparation blood serum sample; Use Precision 2000 then ^TMAutomatically the imbibition system carries out 3 times of serial dilutions to 25 times of initial diluents.The serial dilution for preparing negative control sera (have low-level or background level anti--mice serum of dsDNA antibody) with same procedure.Also can choose the diluent (for example, 5000 of NZB F1 serum times of diluents) of one or more positive control serums of preparation wantonly.

[0250] blood serum sample with dilution adds washed flat board, as adopts and add model machine (rapidplate robot) fast and add the 25ul dilute serum.The at room temperature gentle vibration of flat board was cultivated 2 hours, then with lavation buffer solution washing 6 times.In each hole, add HRP (horseradish peroxidase)-link coupled anti--mice Fc antibody (25 μ l are anti--mu-FcHRP, from Jackson's immune Research laboratory (JacksonImmunoResearch Laboratories of company, Inc.), catalog number (Cat.No.) 115-035-071, with 5000 times of assay buffer dilutions), and with the at room temperature gentle vibration cultivation of flat board 1 hour.Add substrate solution (25 μ l/ holes; A tmb substrate+a peroxidase solution B is all available from K ﹠amp; (the Kirkegaard ﹠amp of P company; Perry)), there is color to occur.Add stop bath (25 μ l/ holes, 1M H ₃PO ₄), at 450/620nm to dull and stereotyped reading.

[0251] calculate the anti-ds-DNA antibody titer of blood serum sample with following formula:

Wherein, CP (cutpoint (cutpoint)) is 3 times of negative control sera absorbance meansigma methods; HighA _450/620Be numerically near but be higher than the absorbance (A of cutpoint _450/620); Low A _450/620Be numerically near but be lower than the absorbance (A of cutpoint _450/620); DF1 be numerically near but be lower than the low A of cutpoint _450/620The dilution gfactor of value; DF2 be numerically near but be higher than the high A of cutpoint _450/620The dilution gfactor of value.

Flow cytometry

[0252] as described below, measure the number that activates the CD4+T cell in the spleen by flow cytometry.Obtain whole spleen, be crushed to single cell suspension, use EL buffer (the molten born of the same parents' buffer of erythrocyte then, available from the proper root (Qiagen of company in Valencia, California city, Valencia, CA), catalog number (Cat.No.) 79217) the dissolving Red blood corpuscle, make it by 70 microns cellular filters, resuspended then to carry out cell counting.(polymerization scientific company (Polysciences, Inc.), catalog number (Cat.No.) 18862) solution mixes with each cell suspension of fixed volume and the fluorescent bead of concentration known.Franklin, New Jersey lake city BD biotechnology company (BD Biosciences, Franklin Lakes, FACScan NJ) that mixture is added then ^TMFlow cytometry.The pearl of the fixed number by collecting each mixture can be calculated the living cells sum, further can determine the cell subsets sum of each mouse spleen after the facs analysis.

[0253] at 1 x 10 ⁶Add the fluorogen-coupling antibody of saturation capacity in the cell, cultivated 30 minutes, then with cold buffer washing on ice.Splenocyte is with resisting-CD4 (BD Fa Ma King Company (BDPharmingen), catalog number (Cat.No.) 553055, clone RM4-4), anti--CD3 (BD Fa Ma King Company, catalog number (Cat.No.) 555276, clone 17A2) and anti--CD69 (BD Fa Ma King Company, catalog number (Cat.No.) 553237 is cloned H1.2F3) dye or dye with anti--CD4, anti--CD3 and anti--CD25 (Mil Te Ni biotech company (Miltenyi Biotec), catalog number (Cat.No.) 130-091-013).CD3 dyeing helps separation of C D4 and cd8 t cell, and this is because cd8 cell is positive to CD3 and CD4 is negative.FACSCalibur with BD biotechnology company ^TMFlow cytometry is by the flow cytometry sample.

Embodiment 2: with non-expendable CD4 Antybody therapy multiple sclerosis

[0254] listed a series of experiments below, proved that non-expendable CD4 antibody is effective in the MS model before clinical.The performance of described antibody is compared with the exemplary criteria product of nursing and experimental treatment.

[0255] experimental allergic encephalomyelitis (EAE) is a kind of and the inflammatory disease similar central nervous system of MS (CNS); In these two kinds of diseases, demyelination all causes the impaired and paralysis of nerve conduction.Efficacy models before providing a kind of useful MS clinical by the recurrence of giving SJL/J injected in mice proteolipid protein(PLP) (PLP) inducing peptide and the EAE that disappears (referring to, for example, Miller and Karpus (1996) " ExperimentalAutoimmune Encephalomyelitis in the Mouse (experimental allergic encephalomyelitis of mice) ", be selected from Current Protocols in Immunology (newly organized immunological experiment guide), Coligan etc. (volume), John Willie father and son company, with Sobel etc., (1990) " Acute experimental allergicencephalomyelitis in SJL/J mice induced by a synthetic peptide of myelinproteolipid protein (SJL/J chmice acute experimental allergic encephalomyelitis of the synthetic inducing peptide of myelin protein lipid protein) " J Neuropathol Exp Neurol.49 (5): 468-79).

[0256] sketch map of Figure 10 for making progress in time to disease behind the injection PLP peptide in this model.Injection in the 0th day, seizure of disease then (0-15 days), (15-25) the also recurrence of disappearing (the 25th day, finished research at 60-70 days).Following allocation criterion clinical neurology scoring: 0-does not have disease; 1-walks lamely, tail or back myasthenia of limbs, but do not occur simultaneously; 2-walks lamely and with tail and back myasthenia of limbs; 3-hind leg partial paralysis; 4-hind leg complete paralysis; Dying with 5-, die from EAE, because the artificial origin is condemned to death.In this sketch map, arrow is illustrated in the time point that begins in the research that Antybody therapy and other treatment are compared with non-expendable CD4 Antybody therapy.Other treatment had shown effective time point before round dot was represented.

[0257] in this model, with effect before non-expendable CD4 antibody clinical and control antibodies (mentioned above), CTLA4-Ig, α-4 alpha 2 integrin antibodies and acetic acid glatiramer (

) compare.The SJL/J mice was the PLP-139-151 peptide immunity with CFA (complete Freund's adjuvant) preparation in the 0th day.As mentioned above, weekly mice is carried out 3 screenings; When terminal point, detect histopathology (brain and spinal cord).If begin treatment after seizure of disease, then the disease score of elder generation's monitoring mice is divided into suitable each group treatment again of disease score then at random.In three independent studies, reach peak value or began antibody (or other) treatment on the 24th day during the disease low ebb in seizure of disease in the 8th day, the 14th day disease.Non-expendable CD4 antibody, control antibodies, CTLA4-Ig, α-4 alpha 2 integrin antibodies and acetic acid glatiramer are sent weekly three times with 10mg/kg.

[0258] except that mentioned above, in these experiments, used non-expendable CD4 antibody is Mus sourceization (murinized) YTS177 antibody.Mus source YTS177 comprises the heavy chain and the variable region of light chain of rat YTS177 antibody, clone's mice IgG2a heavy chain and κ constant region of light chain sequence upstream.Its heavy chain comprises that in the Fc receptor binding domain two monamino acids replace (being changed into alanine with the corresponding residue of N297 with human IgG1's residue D265).

[0259] as shown in figure 11, give (at the 8th day begin treatment) when in seizure of disease, non-expendable CD4 antibody is better than CTLA4-Ig and acetic acid glatiramer.Figure 11 A is the time dependent diagram of clinical score of control antibodies, acetic acid glatiramer, α-4 alpha 2 integrin antibodies, CTLA4-Ig and non-expendable CD4 Antybody therapy group.Figure 11 B has shown average every day of the clinical score of these groups.

[0260] when when the disease peak value gives (at the 14th day begin treatment), described CD4 antibody also is better than CTLA4-Ig, as shown in figure 12.Figure 12 A is the time dependent diagram of clinical score of control antibodies, CTLA4-Ig and described CD4 Antybody therapy group.(acetic acid glatiramer and α-4 alpha 2 integrin antibodies are invalid at this time point.) Figure 12 B shown average every day of the clinical score of these groups.Three independent experiments have been represented with the observed effect of CD4 antibody.

[0261] as shown in figure 13, when when the terminal stage of a disease gives (at the 24th day begin treatment), described CD4 antibody also is better than CTLA4-Ig.Figure 13 A is the time dependent diagram of clinical score of control antibodies, CTLA4-Ig and described CD4 Antybody therapy group.Figure 13 B has shown average every day of the clinical score of these groups.Two independent experiments have been represented with the observed effect of non-expendable CD4 antibody.

[0262] reduced the demyelination of EAE with non-expendable CD4 Antybody therapy, as shown in figure 14.(the 12nd day) begins with antibody (being YTS177 rather than Mus source YTS177 in this experiment) treatment near disease acute phase peak value the time, and continues treatment up to research termination in the 80th day.Collect spinal cord, fixing and dye with the quick blue dyestuff of Luxol (complete myelin can be dyed navy blue).Contour area is the demyelination district.The mice of selecting has been represented the meansigma methods of every group of average demyelination scoring.

[0263] also can reduce the CD4+T cellular infiltration that recurs/disappear the EAE model with CD4 Antybody therapy (Mus source YTS177).For example, 60 animal obtains behind the begin treatment from the 14th day the time, and by above embodiment 1 is described with CD4 and the painted spinal cord slice of CD8, compare with the animal of control antibodies treatment, CD4+ soaks into and reduces in the animal of CD4 Antybody therapy, but CD8+ soaks into reduction.

[0264] mice with non-expendable CD4 Antybody therapy has kept immunocompetence, demonstrates normal survival rate after infect the Listerella.For example, back animal 10/10 survival with non-expendable CD4 antibody (Mus source YTS177) treatment in the time of the 8th day is infected in the Listerella, and with the animal of contrast Ig Antybody therapy have only 8/10, with the animal 3/10 of CTLA4-Ig treatment with animal 0/10 survival (Wooley etc., (1993) the J of Immunol 151 (11): 6602) of TNFRII-Fc treatment.1 day begin treatment before the inoculation Listerella, the initial dose of therapeutic agent is 20mg/kg, the dosage of all therapeutic agents is 5mg/kg 3x/ week in the research afterwards.

[0265] as shown in figure 15, reduce the ratio of CD4+ effect/memory cell in the blood with CD4 Antybody therapy energy selectivity.Shown with ICOS in every microliters of blood of measuring by flow cytometry in the animal of control antibodies, CD4 antibody or CTLA4-Ig treatment ^HiCD4 or ICOS ^HiThe number of CD8 T cell.(ICOS ^HiBe the label of effect/memory T cell, represent in the normal mouse blood T cell, rise to about 15-20% along with EAE produces less than 4%.) being different from CTLA4-Ig, non-expendable CD4 antibody capable reduces the number of CD4+ cell but does not reduce the number of CD8+ cell.In this experiment, treatment was since the 14th day; And at the 46th day pair cell counting.

[0266] in a word, recurring/disappearing in the SJL/J model of EAE effectively with non-expendable CD4 Antybody therapy.Reduce the clinical score of all intervention time points with this Antybody therapy, reduced the histological score of brain and spinal cord, reduced CD4+ among the CNS but not CD8+ soaks into, also reduced ICOS ^HiCD4+ but not the number of CD8+T cell.The effect of this CD4 antibody is higher than CTLA4-Ig and acetic acid glatiramer, and compares favourably with α-4 integrin monoclonal antibody at least.

[0267] with the EAE of CD4 Antybody therapy MOG-inducing peptide in another kind of MS model-C57B1k6 mice-in also effective.MOG model not display cycle property disappears, and more is acute/chronic MS model therefore.When near the begin treatment disease peak value, in the MOG model, to observe neurological symptoms result and reverse rapidly, this is similar with observed phenomenon in the SJL/J model.As shown in figure 16, and compare, reduced clinical score with non-expendable (or expendable) CD4 Antybody therapy with control antibodies, CTLA4-Ig or expendable CD8 Antybody therapy.

Experiment flow

Flow cytometry

[0268] measures the number of effect/memory cell in the blood by following flow cytometry.(retro-orbitally) behind the blood socket of the eye of fixed volume collected in the heparinization pipe, dissolve Red blood corpuscle and resuspended then to carry out cell counting.Each cell suspension with fixed volume mixes to measure the cell subsets sum of each mouse blood, the description in embodiment 1 as mentioned with the fluorescent bead solution of concentration known then.

[0269] at 1 x 10 ⁶Add the fluorogen-coupling antibody of saturation capacity in the cell, cultivated 30 minutes, then with cold buffer washing on ice.Hemocyte is with resisting-CD4 (BD Fa Ma King Company, catalog number (Cat.No.) 553055, clone RM4-4), anti--CD8a (BD Fa Ma King Company, catalog number (Cat.No.) 553033, clone 53-6.7), biotinylation anti--ICOS (BD Fa Ma King Company, catalog number (Cat.No.) 552145, clone 7E.17G9) dyeing, washing then.Hemocyte is used Succ-PEG-DSPE-APC (BD Fa Ma King Company, catalog number (Cat.No.) 554067) dyeing and washing once more then.FACSCalibur with BD biotechnology company passes through the flow cytometry sample.

With Luxol is blue fast spinal cord slice is dyeed

[0270] will be with the quick indigo plant of Luxol with the paraffin-embedded spinal cord slice dyeing of the formalin fixed of 4 μ m section.With spinal cord slice deparaffnize and with 95% hydrous ethanol.Then at 60 ℃ with the fast blue dyeing of Luxol spend the night (at least 16 hours).Fall excess dye with 95% alcohol flushing, and use dH ₂The O washed.By quick submergence 10-20 second in 0.05% lithium carbonate, be replaced by 70% ethanol then several times up to distinguishing that grey matter and white matter distinguish microscope slide.Microscope slide dyeed 5 minutes at 37 ℃ with cresol-purple then, with 95% ethanol rinsing, and slowly dehydration, cleaning is also fixing.Referring to Sheehan (1980) Theory and Practice ofHistotechnology (histotechnology theory and practice), second edition, 263-264 page or leaf.

Infect the Listerella

[0271] gives the listerisa monocytogenes in mjme of inoculating in the mouse vein with 100,000 colony-forming units of 100 microlitre PBS preparation (from ATCC, strain number is 43251).Began through peritoneal injection monoclonal antibody or fusion rotein (every mice 400 μ g are equivalent to 20mg/kg, with 100 μ l PBS preparation) that day before infected the Listerella; After infect the Listerella, continue to give the dosage of time each 100 μ g (5mg/kg) on every Wendesdays, 10 days.The disease sign of twice monitoring every day mice.

Listerial generation

[0272] keeps listerial virulence by continuous passage in the C57B1/6 mice.Obtain the fresh separated thing from the spleen that infects, cultivate with liquid brain heart infusion (BHI) or at BHI agar plate (Di Feike laboratory company (Difco Labs), Detroit, the state of Michigan).With the antibacterial cyclic washing, be resuspended in aseptic PBS and be stored among-80 ℃ the PBS that contains 20% glycerol.

The combined therapy lupus of embodiment 3:CD4 antibody and MMF

[0273] listed a series of experiments below, these experimental results show that independent or effective for the preclinical models of SLE with the non-expendable CD4 antibody of mycophenolate combination.

[0274] the NZB x W F1 mouse model of SLE is as described in the embodiment 1.In this model, with non-expendable CD4 antibody (YTS177, effect and non-binding control antibodies (as mentioned above) before as mentioned above) clinical, mycophenolate (

Or MMF, present therapeutic agent) and the combination of CD4 antibody and MMF compare.

[0275] begin treatment NZB x NZW mice when big at 9 months.The albuminuria situation of root screening mice is according to their albuminuria scoring random packet.During the experiment beginning, each treatment group comprises 15 mices, wherein the 73% albuminuria level that demonstrates〉300mg/dl.(notice that this morbid state is more serious than the morbid state during the treatment beginning in the foregoing description 1 described experiment, has only 32% murine protein urine when the experiment of embodiment 1 begins〉300mg/dl.) mice with contrast Ab, non-expendable CD4 antibody (anti--CD4), MMF (

) or non-expendable anti--CD4 treated 2 months continuously with the combination of MMF.Variation (the embodiment 1 described urinalysis that carries out as mentioned), progression of disease and the survival rate of monitoring murine protein urine.Non-expendable CD4 antibody (YTS177) is sent weekly three times with 5mg/kg by intraperitoneal (IP) injection.MMF gives (making up separately or with CD4 antibody) with 25mg/kg or 50mg/kg IP every day.

[0276] in this experiment, wherein begin treatment when morbid state is serious is not enough to obviously reverse serious albuminuria (some mices improve, but number deficiency is to reach significance) with CD4 antibody or with the MMF treatment separately.Yet, the collaborative remarkable result that produces of combined therapy; As shown in figure 17, observe when giving for reversing albuminuria when CD4 antibody and MMF combination synergy is arranged.Figure 17 A has shown the percentage ratio that indicates the mice of moment albuminuria＜300mg/dl after treatment.With MMF

Combination gives CD4 antibody and causes demonstrating albuminuria〉the mice number of 300mg/dl reduces only, illustrate that the nephritis symptom in the utmost point terminal illness obtains reversing, and this does not observe in the control antibodies or the group for the treatment of with CD4 antibody or MMF separately.Figure 17 B has shown the percentage ratio for the treatment of the mice of reversing afterwards in 1 month from albuminuria 300mg/dl.

[0277] as shown in figure 18, (Figure 18 A and 18B are 50mg/kg/ days under two kinds of MMF dosage, Figure 18 C and 18D are 25mg/kg/ days), the combination that gives non-expendable CD4 antibody and MMF has all postponed the progression of disease time (Figure 18 A and 18C) and improved survival rate (Figure 18 B and 18D).The combination of non-expendable CD4 antibody and MMF is more effective with non-expendable CD4 antibody or MMF than single.In Figure 18 A-18D, reference contrast (control antibodies-treatment group) is represented with black matrix, only expresses the p value of comparing each group with significance,statistical with the reference contrast on figure.

[0278] combined therapy with CD4 antibody and MMF can effectively reduce albuminuria.Figure 19 has shown treatment albuminuretic multiple comparisons analysis 2 months the time, adopt the Dunnett method with control antibodies treatment group as the reference matched group.With the group of 50mg/kg MMF every day (separately or with CD4 antibody combination) treatment the results are shown in Figure 19 A, and the group for the treatment of with 25mg/kg MMF every day (separately or with the combination of CD4 antibody) the results are shown in Figure 19 B.Reference contrast (treating with control antibodies) is represented with black matrix, only expresses the p value of comparing each group with significance,statistical with the reference contrast on figure.The result confirms that the combination of CD4 antibody and MMF provides remarkable benefit for the albuminuria that reduces model, and significant albuminuria alleviates on the statistics and list does not demonstrate with control antibodies, anti--CD4 or MMF treatment.

[0279] combined therapy with non-expendable CD4 antibody and MMF has reduced the number of CD4+T cell in the spleen.Shown in Figure 20 C, compare with the animal of control antibodies treatment, with the spleen CD4 of the bimestrial animal of this combined therapy ⁺The T cell number reduces (p=0.002).For example, in B cell mass and dendritic cell group, also observe the downstream effect of combined therapy.For example, reduced the number of B2B cell in the spleen with CD4 antibody and MMF combined therapy, shown in Figure 20 D (with respect to the animal of control antibodies treatment; P=0.017).Spleen CD4 ⁺The minimizing of T cell and B2B cell be not since antibody consumption this cell, (the seeing Figure 20 A and 20B respectively) that is proved as CD4+T cell in the blood and B2 B TCS.In fact, notice respectively with blood CD4 occurring in the combination of CD4 antibody and 50mg/kg MMF and the group with 25mg/kg MMF treatment ⁺T cell and B2B cell number increase (although these increases may not be that statistics is significant).Substantially as mentioned above measure CD4 by flow cytometry ⁺The number of T cell and B2B cell adopts antibody to identify various cell masses, and the percentage ratio of each colony shown in using then (by total lymphocyte) multiply by lymphocytic sum to determine the number of each cell mass.Identify B2 cell (topmost B cell) by B220 (CD45) and CD38 positive staining.Anti--B220/CD45 and anti-cd 38 are available from BD Fa Ma King Company.

[0280] combined therapy with non-expendable CD4 antibody and MMF has also reduced IgM ⁺The plasma cell number is shown in Figure 20 E.IgM ⁺Plasmacytic number is measured by aforesaid substantially flow cytometry.Plasma cell is identified in expression according to syndecan-1; The IgM plasma cell is also at their syndecan-1 positive cell of surface expression IgM.The antibody of syndecan-1 and IgM is available from BD Fa Ma King Company.Adopt the Dunnett method to compare, wherein control antibodies treatment group is only expressed the p value of comparing each group with significance,statistical with the reference contrast as reference matched group (representing with black matrix) on figure.

[0281] similarly, reduced isotype-change plasmacytic number, shown in Figure 20 F with the combined therapy of CD4 antibody and MMF.The plasmacytic number of isotype-conversion is measured by aforesaid substantially flow cytometry.Plasma cell is identified in expression according to syndecan-1; It is isotype-conversion plasma cell (expressing the isotype outside the IgM, for example IgG, IgE etc.) that IgM expresses syndecan-1 positive cell that is negative.The antibody of syndecan-1 and IgM is available from BD Fa Ma King Company.Adopt the Dunnett method to compare, control antibodies treatment group is only expressed the p value of comparing each group with significance,statistical with the reference contrast as reference matched group (representing with black matrix) on figure.

[0282] also reduced the number of the B of germinal center cell with this combined therapy, shown in Figure 20 G.The number of the B of germinal center cell is measured by aforesaid substantially flow cytometry.Germinal center's B cell is accredited as the B220 positive and cell (this makes them be different from the B2 cell of coexpression B220 and CD38) that the CD38 surface expression is negative.Anti--B220/CD45 and anti-cd 38 are available from BD Fa Ma King Company.Adopt the Dunnett method to compare, control antibodies treatment group is only expressed the p value of comparing each group with significance,statistical with the reference contrast as reference matched group (representing with black matrix) on figure.

[0283] plasma cell sample dendritic cell (Plasmacytoid dendritic cell) are the potential important spin-offs of lupus, and this is because they secrete a large amount of I type interferon (α and interferon-).Therefore it should be noted that with independent or reduced the number of spleen plasma cell sample dendritic cell, shown in Figure 20 H with the CD4 Antybody therapy that MMF makes up.The number of plasma cell sample dendritic cell is measured by aforesaid substantially flow cytometry.Adopt label CD19 and CD3 to get rid of B and T cell respectively; In remaining cell, identify plasma cell sample dendritic cell according to the pDCA expression and the middle expression of their B220 (intermediate expression) of their uniquenesses.Antibody is from BD Fa Ma King Company, but anti--pDCA is from Mil Te Ni biotech company.Adopt the Dunnett method to compare, control antibodies treatment group is only expressed the p value of comparing each group with significance,statistical with the reference contrast as reference matched group (representing with black matrix) on figure.

[0284] in addition, reduced the expression of MHC II class in these dendritic cell with this Antybody therapy (independent or with MMF combination), shown in Figure 20 I.Plasma cell sample dendritic cell adopt at the antibody (Mil Te Ni biotech company) of pDCA by aforesaid substantially flow cytometry and measure, and use at IA ^dAnd IE ^dThe antibody of the general epi-position of MHC II molecule (BD Fa Ma King Company) is assessed their MHC II level.Adopt the Dunnett method to compare, control antibodies treatment group is only expressed the p value of comparing each group with significance,statistical with the reference contrast as reference matched group (representing with black matrix) on figure.Because MHC II level is relevant with the state of activation of these dendritic cell usually, level raises and shows that then the state of activation improves, and therefore this observed result explanation can reduce this dendritic cell group's the state of activation with the CD4 Antybody therapy.

[0285] in a word, for example effective for NZB x WF1 mice with non-expendable CD4 Antybody therapy with the MMF combination, even if introduce in the terminal stage of a disease.With time and the survival rate that this combined therapy has prolonged no disease progression, reduced the number of spleen CD4+T cell.In addition, the albuminuria aspect that non-expendable CD4 antibody and MMF is combined in the NZB/W F1 model that reverses SLE obviously is better than singly using MMF.Single with non-expendable CD4 antibody also can the selectivity minimizing B of germinal center cell and isotype-change plasmacytic number, but topmost B cell (B2 cell) is had no significant effect.In addition, anti--CD4 can reduce the number of plasma cell sample dendritic cell, and this cell is relevant with the pathogenesis of SLE with β with IFN-α because of producing 1 type interferon.

[0286] although for clarification and the purpose understood than having described foregoing invention in greater detail, but those skilled in the art obviously should be understood that under the situation that does not deviate from validation range of the present invention and can make various changes aspect form and the details by reading these contents.For example, all above-mentioned technology and compositions can variously be used in combination.Including this paper in is used for all purposes in full by reference for all publications of quoting among the application, patent, patent application and/or other documents, and its degree is as including in for all purposes publication that each is independent, patent, patent application and/or other documents.

Claims (40)

1. method for the treatment of the lupus of mammalian object, described method comprises: the non-expendable CD4 antibody and at least a second combination of compounds of organizing that is selected from down that give described object treatment effective dose: cyclophosphamide, mycophenolate and CTLA4-Ig.

2. the method for claim 1 is characterized in that, and is described to liking the people.

3. the method for claim 1 is characterized in that, described second chemical compound is a cyclophosphamide.

4. the method for claim 1 is characterized in that, described antibody comprises the CDR with aminoacid sequence shown in the SEQ ID NO:27.

5. the method for claim 1 is characterized in that, described antibody comprises the CDR with aminoacid sequence shown in the SEQ ID NO:30.

6. the method for claim 1 is characterized in that, described antibody comprises the CDR with aminoacid sequence shown in SEQ ID NO:25, SEQ ID NO:26 and the SEQ ID NO:27.

7. the method for claim 1 is characterized in that, described antibody comprises the CDR with aminoacid sequence shown in SEQ ID NO:28, SEQ ID NO:29 and the SEQ ID NO:30.

8. the method for claim 1 is characterized in that, described antibody comprise have SEQ ID NO:25, the CDR of aminoacid sequence shown in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and the SEQID NO:30.

9. the method for claim 1, it is characterized in that described antibody comprises heavy chain amino acid sequence shown in light-chain amino acid sequence shown in heavy chain amino acid sequence shown in light-chain amino acid sequence shown in heavy chain amino acid sequence, the SEQ ID NO:15 shown in light-chain amino acid sequence shown in heavy chain amino acid sequence, the SEQ ID NO:9 shown in light-chain amino acid sequence shown in the SEQ ID NO:3 and the SEQ ID NO:6 and the SEQ ID NO:12 and the SEQ ID NO:18 or the SEQ ID NO:21 and the SEQ ID NO:24.

10. method as claimed in claim 9 is characterized in that, described is cyclophosphamide to liking people and wherein said second chemical compound.

11. method as claimed in claim 10 is characterized in that, described lupus is a lupus nephritis.

12. method as claimed in claim 11 is characterized in that, described lupus is II level lupus nephritis, III level lupus nephritis, IV level lupus nephritis or V level lupus nephritis.

13. method as claimed in claim 11 is characterized in that, behind described combination begin treatment, described object shows that albuminuria alleviates and/or the activeness urinary sediment alleviates.

14. the method for claim 1, it is characterized in that described antibody comprises the CD4 binding fragment of the antibody that contains following light-chain amino acid sequence and heavy chain amino acid sequence: heavy chain amino acid sequence shown in light-chain amino acid sequence shown in the SEQ ID NO:3 and the SEQ ID NO:6, heavy chain amino acid sequence shown in light-chain amino acid sequence shown in the SEQ ID NO:9 and the SEQ ID NO:12, heavy chain amino acid sequence shown in light-chain amino acid sequence shown in the SEQ ID NO:15 and the SEQ ID NO:18, or heavy chain amino acid sequence shown in light-chain amino acid sequence shown in the SEQ ID NO:21 and the SEQ ID NO:24.

15. the method for claim 1, it is characterized in that, described antibody is and the bonded CD4 antibody of identical epi-position that is selected from down the antibody of organizing: the antibody that comprises heavy chain amino acid sequence shown in light-chain amino acid sequence shown in the EQ ID NO:3 and the SEQID NO:6, the antibody that comprises heavy chain amino acid sequence shown in light-chain amino acid sequence shown in the SEQ ID NO:9 and the SEQ ID NO:12, comprise the antibody of heavy chain amino acid sequence shown in light-chain amino acid sequence shown in the SEQ ID NO:15 and the SEQ ID NO:18 and comprise light-chain amino acid sequence shown in the SEQ IDNO:21 and SEQ ID NO:24 shown in the antibody of heavy chain amino acid sequence.

16. the method for claim 1 is characterized in that, described antibody is humanized antibody.

17. the method for claim 1 is characterized in that, described antibody has nonglycosylated Fc part.

18. the method for claim 1 is characterized in that, described antibody is not in conjunction with the Fc receptor.

19. the method for claim 1, it is characterized in that, described antibody comprises aminoacid replacement on the one or more positions in Fc district amino acid position 270,322,326,327,329,313,333 and/or 334, and this replacement has changed the C1q combination and/or depended on the cytotoxicity of complement.

20. the method for claim 1 is characterized in that, described antibody comprises remedies the receptors bind epi-position.

21. the method for claim 1 is characterized in that, described antibody comprises the serum albumin binding peptide.

22. the method for claim 1 is characterized in that, described antibody comprises three or more antigen binding site.

23. the method for claim 1 is characterized in that, described lupus is a systemic lupus erythematosus (sle).

24. the method for claim 1 is characterized in that, described lupus is a lupus erythematosus,cutaneous.

25. the method for claim 1 is characterized in that, described lupus is a lupus nephritis.

26. method as claimed in claim 25 is characterized in that, described lupus is II level lupus nephritis, III level lupus nephritis, IV level lupus nephritis or V level lupus nephritis.

27. method as claimed in claim 25 is characterized in that, behind described combination begin treatment, described object shows that albuminuria alleviates and/or the activeness urinary sediment alleviates.

28. the method for claim 1 is characterized in that, before described combination begin treatment, described object demonstrates albuminuria, has improved this albuminuria by treatment.

29. the method for claim 1 is characterized in that, behind described combination begin treatment, lupus improves; Described method comprises, observes after this improvement, stops with described combined therapy object and gives the described non-expendable CD4 antibody of described object treatment effective dose.

30. the method for claim 1 is characterized in that, behind described combination begin treatment, lupus improves; Described method comprises, observes after this improvement, stops with described combined therapy object and gives described second chemical compound or one or more other chemical compounds of described object treatment effective dose.

31. method for the treatment of the lupus nephritis of mammalian object, described method comprises: the non-expendable CD4 antibody that gives described object treatment effective dose, wherein, show that with described object behind the described antibody begin treatment renal function improves, albuminuria alleviates and/or the activeness urinary sediment alleviates.

32. method as claimed in claim 31 is characterized in that, and is described to liking the people.

33. method as claimed in claim 32, it is characterized in that, before described antibody begin treatment, described object shows that albuminuria is higher than 500mg/ days, is higher than 1000mg/ days, is higher than 2000mg/ days or is higher than 3500mg/ days, reduces with this albuminuria behind the described antibody begin treatment.

34. method as claimed in claim 31 is characterized in that, described antibody is selected from down group:

A) comprise the antibody of heavy chain amino acid sequence shown in light-chain amino acid sequence shown in the SEQ ID NO:3 and the SEQ ID NO:6;

B) comprise the antibody of heavy chain amino acid sequence shown in light-chain amino acid sequence shown in the SEQ ID NO:9 and the SEQ ID NO:12;

C) comprise the antibody of heavy chain amino acid sequence shown in light-chain amino acid sequence shown in the SEQ ID NO:15 and the SEQ ID NO:18;

D) comprise the antibody of heavy chain amino acid sequence shown in light-chain amino acid sequence shown in the SEQ ID NO:21 and the SEQ ID NO:24;

E) comprise a), b), c) or the antibody of the CD4 binding fragment of antibody d);

F) comprise antibody with CDR of aminoacid sequence shown in the SEQ ID NO:27;

G) comprise antibody with CDR of aminoacid sequence shown in the SEQ ID NO:30;

H) comprise antibody with CDR of aminoacid sequence shown in SEQ ID NO:25, SEQ ID NO:26 and the SEQ ID NO:27;

I) comprise antibody with CDR of aminoacid sequence shown in SEQ ID NO:28, SEQ ID NO:29 and the SEQ ID NO:30; With

J) comprise have SEQ ID NO:25, the antibody of the CDR of aminoacid sequence shown in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and the SEQ ID NO:30.

35. method as claimed in claim 31, it is characterized in that, described antibody is and the bonded CD4 antibody of identical epi-position that is selected from down the antibody of organizing: the antibody that comprises heavy chain amino acid sequence shown in light-chain amino acid sequence shown in the EQ ID NO:3 and the SEQID NO:6, the antibody that comprises heavy chain amino acid sequence shown in light-chain amino acid sequence shown in the SEQ ID NO:9 and the SEQ ID NO:12, comprise the antibody of heavy chain amino acid sequence shown in light-chain amino acid sequence shown in the SEQ ID NO:15 and the SEQ ID NO:18 and comprise light-chain amino acid sequence shown in the SEQ IDNO:21 and SEQ ID NO:24 shown in the antibody of heavy chain amino acid sequence.

36. method as claimed in claim 31 is characterized in that, described lupus is II level lupus nephritis, III level lupus nephritis, IV level lupus nephritis or V level lupus nephritis.

37. a method for the treatment of the disease of mammalian object, described method comprises:

Give the non-expendable CD4 antibody and at least a second combination of compounds that is selected from down group of described object treatment effective dose: cyclophosphamide, mycophenolate and CTLA4-Ig;

Wherein, described disease is selected from down group: rheumatoid arthritis, asthma, psoriasis, transplant rejection, graft versus host disease, multiple sclerosis, Crohn disease, ulcerative colitis and xerodermosteosis.

38. method as claimed in claim 37 is characterized in that, described disease is selected from down group: rheumatoid arthritis, asthma, psoriasis, transplant rejection and graft versus host disease.

39. method as claimed in claim 37 is characterized in that, and is described to liking the people.

40. method as claimed in claim 37 is characterized in that, described antibody is selected from down group:

A) comprise the antibody of heavy chain amino acid sequence shown in light-chain amino acid sequence shown in the SEQ ID NO:3 and the SEQ ID NO:6;

B) comprise the antibody of heavy chain amino acid sequence shown in light-chain amino acid sequence shown in the SEQ ID NO:9 and the SEQ ID NO:12;

C) comprise the antibody of heavy chain amino acid sequence shown in light-chain amino acid sequence shown in the SEQ ID NO:15 and the SEQ ID NO:18;

D) comprise the antibody of heavy chain amino acid sequence shown in light-chain amino acid sequence shown in the SEQ ID NO:21 and the SEQ ID NO:24;

E) comprise a), b), c) or the antibody of the CD4 binding fragment of antibody d);

F) comprise antibody with CDR of aminoacid sequence shown in the SEQ ID NO:27;

G) comprise antibody with CDR of aminoacid sequence shown in the SEQ ID NO:30;

H) comprise antibody with CDR of aminoacid sequence shown in SEQ ID NO:25, SEQ ID NO:26 and the SEQ ID NO:27;

I) comprise antibody with CDR of aminoacid sequence shown in SEQ ID NO:28, SEQ ID NO:29 and the SEQ ID NO:30; With

J) comprise have SEQ ID NO:25, the antibody of the CDR of aminoacid sequence shown in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and the SEQ ID NO:30.

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