CN101015693A - Use of CD25 binding molecules in steroid-resistant patients. - Google Patents

Abstract

A method for the treatment of a disease selected from the group consisting of autoimmune hepatitis, eczema, vasculitis, temporal arteritis, sarcoid and Crohn's disease, in a steroid-resistant or steroid-sensitive patient, comprising administering to the patient an effective amount of a CD25 binding molecule.

Description

The purposes of CD25 binding molecule in steroid-resistant patients

The application is the denomination of invention submitted on April 5th, 2002 dividing an application for the International Application PCT/EP0203808 of " purposes of CD25 binding molecule in steroid-resistant patients ", described international application entered the China national stage on 27th in JIUYUE in 2003, and application number is 02807480.7.

Technical field

The present invention relates to the CD25 binding molecule and treat the purposes that is selected from following disease in steroid-resistant patients, described disease is selected from: autoimmune hepatitis, asthma, eczema, vasculitis, the property a crossed arthritis, systemic lupus erythematosus (sle), leukemia, glomerulonephritis, multiple sclerosis, ulcerative colitis, sarcoidosis and segmental enteritis.

Background technology

Ulcerative colitis

Concerning great majority suffered from the patient of ulcerative colitis, steroid was a kind of effective therapeutic modality.Yet the patients of ulcerative colitis of 30%-50% is invalid to steroid therapy, and these patients' medical treatment is also undesirable, and Most patients needs operative treatment.Before steroid being used for serious ulcerative colitis treatment, this sick mortality rate is about 50% (Truelove SC﹠amp; Witts LJ, BMJ 1955; Ii:1041-8).At present, clinical way is that ineffective serious patients of ulcerative colitis behind 5-7 days steroid therapies is implemented surgical operation (Jewell DP etc., Gastroenterol Int 1991 immediately; 4:161-4).Although surgical operation can be cured fully, owing to relate to ileostomy (being temporary at least), thereby patient's surgical operation of usually being unwilling to accept.Yet postponing operation is breakneck for the invalid patient of those medical treatments because may perform the operation preceding perforation, and cause thus increasing dead probability (Ritchie JK, BMJ 1974; I:264-268).As the alternative medicine of operation, the immunosuppressant cyclosporin has been applied to treat patients of ulcerative colitis (Lichtiger S etc., the N Engl J Med 1994 of steroid tolerance type; 330:1841-5).Yet, long-term results is disappointed, the patient initially can be effective, but still the treatment that need in six months, undergo surgery, part patient is then owing to immunosuppressant causes fatal opportunistic infection and renal failure and hypertension (Smith MB, N Engl J Med 1992 as cassette pneumocystis carinii pneumonia and so on; 327:497-8; Sandborn WJ, Inflamm Bowel Dis 1995; 1:48-63).Azathioprine is a kind of effective Therapeutic Method to the ulcerative colitis of chronic active sex steroid tolerance type or steroid dependent form, yet its onset cycle long (3-4 month), be not suitable for serious patients of ulcerative colitis (Ardixxone S etc., JClin Gastroenterol 1997 that those are badly in need of treatment so mean it; 25:330-3).Therefore, the treatment of steroid tolerance type ulcerative colitis is still very difficult, often need cause the surgical operation of wound.So, the method that needs other treatment steroid tolerance type ulcerative colitis, and need be interpreted as the reason what some patients of ulcerative colitis is invalid to steroid therapy, concerning discrete patient, predicting it, whether steroid therapy to be produced reaction be very difficult, therefore needs operative treatment or other medical treatment method.In serious ulcerative colitis, the initial order of severity of disease and last result (steroid therapy effect) relation little (Lindgren SC etc., Eur J Gastrenterol Hepatol, 1998; 10:831-5), therefore, the not necessarily the most serious patient of patient that those are invalid to steroid therapy.

Asthma

At present, the method for treatment asthma is for giving the steroid medicine, as glucocorticoid.Although most of patient reaction usually is good, it is invalid to Steroid treatment always to have some patients.These patients are called as and suffer from " steroid tolerance type asthma ", and are found first in nineteen sixty-eight.Subsequently, steroid tolerance type asthma is defined as follows: with every day 20mg dosage give prednisone after 7 days, can not be with per second forced expiration volume (FEV ₁The index that pulmonary function reduces during asthma), and raising 15% (Carmichael J etc., BMJ 1981; 282:1419-22).Steroid is tolerated the type asthmatic patient and the steroid sensitaive asthma patient compares, find they basic pulmonary function test result and they to the reactive and there was no significant difference of bronchiectasis medicine, this shows that steroid tolerance type asthma does not have and there is no more serious disease.Stimulate lymphocytosis suppressed by glucocorticoid according to lectins (PHA), can measure sensitivity (Poznansky MC etc., the Clin Sci 1984 of the lymphocyte of steroid tolerance type asthmatic patient peripheral blood lymphocyte steroid; 67:639-45) (Alvarez etc., J Allergy Clin Immunol 1992; 89:714-21) (CorriganCJ etc., Am Rev Respir Dis 1991; 144:1026-32) (Hackzu etc., J Allergy ClinImmunol 1994; 93:510-9) (Spahn JD etc., J Allergy Clin Immunol 1996; 98:1073-9).All these researchs all show, steroid suppresses to be markedly inferior to lymphocytic inhibition to the steroid sensitaive asthma patient to the lymphocyte of steroid tolerance type asthmatic patient, shows that steroid tolerance type asthmatic patient has steroid tolerance type lymphocyte.Further comprising eight steroid tolerance type asthmatic patients in the research, find that they all have tolerance type lymphocyte, treat (Vrugt B etc., Eur Respir J 2000 with methotrexate to it; 15:478-85).After eight weeks, find that bronchial mucosal inflammation reduces, lymphocyte increases steroid sensitivity.The good efficacy of methotrexate in treatment steroid sensitaive asthma patient bronchial mucosa be since its to have improved due to the sensitivity of steroid this viewpoint still disputable.This is medicine as " steroid reinforcing agent ", increases lymphocyte to the sensitivity of steroid and an example of clinical efficacy is arranged.About 25% patient in " refractory " type asthma (refers to that one three property centers (a tertiary referral centre) have been found steroid tolerance type lymphocyte (Chan MTS etc., J Allergy clin Immunol 1998; 101:594-601).

Autoimmune hepatitis

Autoimmune hepatitis is to be a kind of chronic hepatopathy form of feature with carrying out property hepatocyte inflammation, and is effective usually during with corticosteroid treatment.Yet, still have 10% autoimmune hepatitis patient invalid, and then develop into and carry out hepatopathy and liver cirrhosis corticosteroid treatment.These steroid-resistant patients treatment of diseases are difficult, may need to transplant.Cyclosporin has been used as medicine (Fernandes NF etc., the Am J Gastroenterol1999 of treatment steroid tolerance type autoimmune hepatitis; 94:241-8).In the test that 5 patients are carried out, 4 patient treatment successes.Unfortunately, the patient that this unique example is failed to respond to any medical treatment to cyclosporin develops into carrying out property liver failure, through normal position liver transplantation, dies from the dissemination cytomegalovirus infection at last.Steroid tolerance type autoimmune hepatitis patient has steroid tolerance type lymphocyte according to estimates.

Eczema

Eczema is by multiple external environment conditions such as for example contactant and stimulus object, infectant and specificity allergy or the scytitis with characteristic morphology that internal factor caused or caused.Most of eczema patients symptom is lighter, only needs intermittently topical therapeutic.Yet part patient shows as more serious pathological changes, therefore, needs systematic steroid therapy (Hoare C etc., HealthTechnol Assess 2000; 4:1-191) (Heddle RJ etc., BMJ (Ciin Res Ed) 1984; 289:651-4).To the invalid needs of patients of steroid immunosuppressive drug such as cyclosporin therapy (Granlund H etc., Acta derm.Venereol.1996 as an alternative; 76:371-6).According to estimates, invalid to steroid therapy is because steroid-resistant patients has due to the steroid tolerance type lymphocyte.

Vasculitis

It is the pathological changes of feature with the arterial inflammation that vasculitis is one group.Part in these pathological changes also causes glomerulonephritis (comprising Wegner granulomatosis, Goodpasture and polyarteritis nodosa joint).Steroid be still these pathological changes the first-line treatment medicine (Bosch X etc., Lupus 1999; 8:258-62) (Guillevin L nephrol.Dial.Transplant 199; 14:2077-9) (Reinhold-Keller E etc., Arthritis Rheum.2000; 43:1021-32).The immunosuppressant agent therapy uses with Steroid treatment usually, if this conjoint therapy is invalid, will be fatal.It is believed that the patient who fails to respond to any medical treatment in a large number has steroid tolerance type lymphocyte.

One property crossed arthritis

One property crossed arthritis also is vasculitic a kind of form.Its complication is to disturb the blood supply of eyes to cause that about 15% patient is to (Chevalet P etc., the J Rheumatol 2000 of failing to respond to any medical treatment of a line medicine steroid by arterial inflammation for blind; 27:1484-91).These patients grow the invalid steroid therapy course of treatment usually, and alternative medicine such as methotrexate have been used to treat this class steroid tolerance type disease (Krall PL etc., Cleve Clin J Med 1989; 56:253-7).According to estimates, invalid to steroid therapy is because steroid-resistant patients has due to the steroid tolerance type lymphocyte.

Sarcoidosis

Sarcoidosis is a kind of systemic granuloma disorder of unknown cause.Sarcoidosis is to cause owing to unknown antigen is produced the cell-mediated excessive immunoreation of T.Steroid is still first-line treatment medicine (Barnard J etc., Curr Opin Rheumatol 2001; 13:84-91).Because the intolerance of steroid or intention reduce the dosage and the side effect of steroid, usually adopt the substitute of steroid.

Systemic lupus erythematosus (sle)

Systemic lupus erythematosus (sle) is the over-drastic a kind of autoimmune system disorders of B-cell reactivity that comprises that the T lymphocyte is unusual and produce autoantibody.If do not treat, this disease can cause latter stage nephropathy and death.

Glucocorticoid is the choice drug of therapy system lupus erythematosus.Yet some patients are invalid to steroid therapy.

External (Seki M etc., the Arthritis Rheum 1998 of in systemic lupus erythematosus (sle) (SLE) patient, measuring of dependency between the effectiveness of lymphocyte steroid sensitivity and clinical steroid therapy; 41:823-30).The Japanese women of oral prednisone (every day 50-60mg, continue one month) that studied 27.According to the clinical efficacy of steroid, divide 19 patients into the steroid responsive type, 8 patients divide steroid tolerance type into.Measure lymphocyte to steroid sensitivity by measuring the inductive peripheral blood lymphocyte apoptosis of external steroid (cell death).The lymphocyte of Patients with SLE is in the meticortelone of high concentration, and steroid tolerance type group has significantly low cell death percentage ratio (being respectively 44.8% and 68.8%, p＜0.05) with respect to steroid responsive type group.Therefore, the method of steroid sensitivity is different from detects glucocorticoid and suppress lectins and stimulate lymphoproliferative method although detect lymphocyte, yet find that still the invalid patient of steroid therapy is had steroid tolerance type lymphocyte.

Leukemia

Leukemia is with leukocyte precursor sample hypertrophy and is not divided into the pernicious leukocyte disorder of feature.Although leukemia is not an inflammatory lesion,, glucocorticoid still is included in the chemotherapy scheme of this pathological changes of treatment.Glucocorticoid is induced the lymphoblast apoptosis, and this can be as detecting the index of acute lymphoblast leukemia (ALL) to steroid sensitivity.External high apoptosis rate is relevant with good clinical efficacy.On the contrary, about 30% the steroid toleration takes place in the lymphoblast leukemia case of new diagnosis, and the steroid toleration is that (Kaspers GJL is etc., Leuk Lymphoma 1994 for a serious clinical problem; 13:187-201).In external two researchs carrying out, not only compared lymphocyte to steroid (dexamethasone and meticortelone) sensitivity, and compared sensitivity to a left-handed asparaginase and vincristine (KaspersGJL, etc., Blood 1997; 90:2723-9) (Hongo T etc., Blood 1997; 89:2959:65).With lymphocyte to the sensitivity of these medicines as index, the patient is divided into responsive type (to all medicaments insensitives), moderate type (to two or three kind of medicaments insensitive) and tolerance type (none medicaments insensitive or to a kind of medicaments insensitive).In the responsive type patient, the survival rate when not ill was the highest (85-100%) in 3 years, and resistant patients minimum (43-55%).Therefore, in acute lymphoblast leukaemic, the sensitivity of the extracorporeal sensitivity of lymphoblast and the interior disease of body is not only with relevant to the sensitivity of steroid but also also relevant with other chemotherapeutant.

Glomerulonephritis

Glomerulonephritis is feature, normally autoimmune disease with the kidney inflammation.Steroid is that (Austin HA waits N Engl J Med 1986 to important medicine; 314:614-9), steroid therapy is invalid, and this pathological changes can cause renal failure.Patient with the invalid steroid tolerance type nephropathy of steroid therapy immunosuppressant class medicine (as described in the ulcerative colitis part) (McCauley J etc., Nephrol Dial Transplant 1993 have now been attempted using; 8:1286-90).The lymphocyte that studies show that carries out in 16 glomerulonephritis patients there are differences to steroid sensitivity that (Briggs WA is etc., J Clin Pharmacol 1996; 36:707-14).Although to lymphocyte to the steroid sensitivity difference with to the different explanation of the curative effect of steroid therapy on have dispute, yet in this research, clinical efficacy and lymphocyte are not contrasted steroid sensitivity.FSG (FSGS) is brightic a kind of form, and it also can use steroid therapy, and (Ingulli E is etc., J Am SocNephrol 1995 although the part patient is of no curative effect; 5:1820-5).In this disease, lymphocyte is contrasted steroid sensitivity and clinical efficacy, demonstrate when using steroid therapy, renal function is improved.The patient of the responsive type of lymphocyte is best with the curative effect of steroid therapy, and those have the lymphocytic patient of steroid tolerance type the poorest with the steroid curative effect.

Multiple sclerosis

Multiple sclerosis is the inflammatory disorders of central nervous system's demyelination, with myelin protein interactional T lymphocyte takes place and plays an important role in causing a disease.(Brusaferri F is etc., J Neurol.2000 can to adopt glucocorticoid treatment multiple sclerosis; 247:435-42).Steroid therapy is effective to the rehabilitation in a short time of quickening multiple sclerosis patients, also can effectively reduce the probability of recurrence.Yet in different patients, clinical efficacy is obviously different.In multiple sclerosis patients, also measured lymphocyte to steroid sensitivity (Wei T etc., Acta NeurologicaScandinavica 1997; 96:28-33), find that lymphocyte sensitivity has a long way to go.Clinical efficacy and the lymphocyte also do not studied the steroid therapy multiple sclerosis are contrasted steroid sensitivity, but to external lymphocyte to the steroid sensitivity difference can partial interpretation why the steroid curative effect there are differences disputable.

Lymphocyte is to the sensitivity of steroid

As mentioned above, lymphocyte can be stimulated and hypertrophy (Lindahl Kiessling K, Peterson RD.Exp Cell Res 1969 by the mitogen lectins external; 5585-7).In culture medium, add steroid and can suppress lymphocytosis (Walker KB etc., Transplant1985; 17:1676-8).Under the part situation, lymphocyte is the steroid responsive type, adds micro-steroid and produces tangible hypertrophy inhibitory action, and under the part situation, lymphocyte is a steroid tolerance type, also only produces small hypertrophy inhibitory action even add a large amount of steroid.The inventor in the past after deliberation the dependency between the clinical efficacy of the lymphocyte steroid sensitivity of one group of serious patients of ulcerative colitis and steroid therapy (Hearing SD etc., Gut 1999; 45:382-8).The equal intravenous of all patients gives the steroid of standard dose, suppresses the peripheral blood lymphocyte hypertrophy of lectins stimulation according to dexamethasone and measures the sensitivity of patient's lymphocyte to steroid.There is significant correlation in proof between clinical efficacy and lymphocyte steroid sensitivity.It is all effective to steroid therapy that all that has the lymphocytic patient of steroid responsive type, and all that has steroid tolerance type lymphocyte patient then to the steroid therapy weak effect.Therefore, in ulcerative colitis, lymphocyte has steroid tolerance type lymphocyte and is indicating that clinical efficacy is poor having dependency between the sensitivity of steroid and the steroid clinical efficacy.

Interleukin is the one group of cytokine that is produced by activated macrophage and lymphocyte in immunoreation.They act on lymphocyte and other leukocyte, stimulate their hypertrophy and differentiation.Interleukin II (IL-2) is a kind of of interleukin, and it is one of cytokine that is produced at first by activated T cells, and it stimulates T hyperplasia and the differentiation that produces it in the mode of autocrine.In ulcerative colitis, the interleukin II that activated T lymphocyte produces has play a part important in the process that makes the mucosa inflammation, it is to produce more cytokine by activating other inflammatory cell, thereby produce (Kirman I etc., J.Dig DisSci 1995 due to the raising and activate of more inflammatory cell; 40:291-5).Suppose that a patient is effective to steroid therapy, the important function of steroid is the generation (steroid has multiple effect, suppresses the interleukin II Just One Of Those Things) that suppresses the generation of interleukin II and stop lymphocytosis and related inflammation so.Yet if the patient is a steroid tolerance type, steroid can not suppress the generation of interleukin II so, can not the inflammation-inhibiting process.

In view of the patient who suffers from autoimmune hepatitis, asthma, eczema, vasculitis, the property a crossed arteritis, systemic lupus erythematosus (sle), leukemia, glomerulonephritis, multiple sclerosis, ulcerative colitis, sarcoidosis, segmental enteritis in a large number uses steroid therapy invalid, so need a kind of effective Therapeutic Method to substitute urgent, invasive therapeutic method of surgery.

Summary of the invention

More specifically, aspect first, the invention provides CD25 binding molecule or its equivalent purposes in treatment autoimmune hepatitis, asthma, eczema, vasculitis, the property a crossed arthritis, systemic lupus erythematosus (sle), leukemia, glomerulonephritis, multiple sclerosis, ulcerative colitis, sarcoidosis and segmental enteritis, the CD25 binding molecule comprises at least one antigen binding site, this binding site comprises at least one domain, and described domain comprises hypervariable region CDR1, CDR2 and CDR3 successively; The aminoacid sequence of described CDR1 is Arg-Tyr-Trp-Met-His, the aminoacid sequence of described CDR2 is Ala-Ile-Tyr-Pro-Gly-Asn-Ser-Asp-Thr-Ser-Tyr-Asn-Gln-Lys-Phe-Glu-Gly, and the aminoacid sequence of described CDR3 is Asp-Tyr-Gly-Tyr-Tyr-Phe-Asp-Phe.Show in the research as following embodiment 1 and embodiment 2 that CD25 binding molecule of the present invention can be regulated the sensitivity of lymphocyte to steroid, so that steroid-resistant patients is transformed into steroid responsive type patient.Therefore, CD25 binding molecule of the present invention can be used for the treatment of autoimmune hepatitis, asthma, eczema, vasculitis, the property a crossed arthritis, systemic lupus erythematosus (sle), leukemia, glomerulonephritis, multiple sclerosis, ulcerative colitis, sarcoidosis and the segmental enteritis of steroid-resistant patients.

And CD25 binding molecule of the present invention also can be used to improve the curative effect of steroid sensitivity patient to steroid therapy.Although expection steroid responsive type patient is effective to steroid therapy, in any case but, treat with CD25 binding molecule of the present invention that this type of patient is taken effect is faster, perhaps can select the more steroid of low dosage for use, thereby can significantly reduce the side effect of steroid therapy.Therefore, according to another aspect of the present invention, also provide the method for prevention, alleviation/prevention of recurrence and the following disease of treatment steroid responsive type patient: chronic active hepatitis, asthma, eczema, vasculitis, the property a crossed arthritis, systemic lupus erythematosus (sle), leukemia, glomerulonephritis, multiple sclerosis, ulcerative colitis, sarcoidosis, segmental enteritis, described method comprises the CD25 binding molecule of the present invention that gives described patient's effective dose, and gives steroid where necessary.

" CD25 binding molecule " thus be meant and anyly can form the molecule of height affinity interleukin II receptor in conjunction with CD25 antigen itself or with the bonded CD25 antigen of other molecule.

Preferred CD25 binding molecule or its direct equivalent that comprises at least one antigen binding site that adopt, this binding site comprises:

A) comprise first domain of CDR1, CDR2 and CDR3 hypervariable region successively; The aminoacid sequence of described CDR1 is Arg-Tyr-Trp-Met-His, the aminoacid sequence of described CDR2 is Ala-Ile-Tyr-Pro-Gly-Asn-Ser-Asp-Thr-Ser-Tyr-Asn-Gln-Lys-Phe-Glu-Gly, and the aminoacid sequence of described CDR3 is Asp-Tyr-Gly-Tyr-Tyr-Phe-Asp-Phe

B) comprise second domain of CDR1 ', CDR2 ' and CDR3 ' hypervariable region successively, the aminoacid sequence of described CDR1 is Ser-Ala-Ser-Ser-Ser-Ile-Ser-Tyr-Met-Gln, the aminoacid sequence of described CDR2 ' is Asp-Thr-Ser-Lys-Leu-Ala-Ser, and the aminoacid sequence of described CDR3 ' is His-Gln-Arg-Ser-Ser-Tyr-Thr;

Except that specializing, any polypeptide chain described here all refers to the terminal beginning of N-, with the terminal aminoacid sequence that finishes of C-.

When antigen binding site comprises first and second domains, these domains can be positioned at same peptide molecule, perhaps preferably each domain is positioned on the different peptide chains, first domain is as heavy chain immunoglobulin or its segmental part, and the mat woven of fine bamboo strips two domains are as a light chain immunoglobulin or a segmental part.

Therefore, the present invention also provides the purposes of the CD25 binding molecule that comprises at least one antigen binding site, described binding site comprises first domain and second domain, the aminoacid sequence of first domain and EP 449, Seq.Id.No.1 shown in the 769B1 is identical or basic identical, begin and finish or first domain is first domain as indicated above by 1 amino acids with 117 amino acids, the aminoacid sequence of second domain and EP 449, Seq.Id.No.2 shown in the 769B1 is identical or basic identical, begin and finish by 1 amino acids with 104 amino acids, EP 449, and the content of 769B1 is incorporated herein for referencial use at this.

According to the present invention, preferred CD25 binding molecule is selected from chimeric anti-CD 25 antibody and its direct equivalent, and this antibody comprises at least:

A) heavy chain immunoglobulin or its fragment comprise that (i) comprises the variable domains of CDR1, CDR2 and CDR3 hypervariable region and (ii) people's heavy chain constant portion or its fragment successively; The aminoacid sequence of described CDR1 is Arg-Tyr-Trp-Met-His, the aminoacid sequence of described CDR2 is Ala-Ile-Tyr-Pro-Gly-Asn-Ser-Asp-Thr-Ser-Tyr-Asn-Gln-Lys-Phe-Glu-Gly, the aminoacid sequence of described CDR3 be Asp-Tyr-Gly-Tyr-Tyr-Phe-Asp-Phe and

B) light chain immunoglobulin or its fragment comprise that (i) comprises the variable domains of CDR1 ', CDR2 ' and CDR3 ' hypervariable region and (ii) constant portion or its fragment of people's light chain successively; The aminoacid sequence of described CDR1 ' is Ser-Ala-Ser-Ser-Ser-Ile-Ser-Tyr-Met-Gln, the aminoacid sequence of described CDR2 ' is Asp-Thr-Ser-Lys-Leu-Ala-Ser, and the aminoacid sequence of described CDR3 ' is His-Gln-Arg-Ser-Ser-Tyr-Thr.

According to the present invention, the strand binding molecule of the also optional self-contained antigen binding site of CD25 binding molecule and its direct equivalent, described binding site comprises

A) comprise first domain that CDR1, CDR2 and CDR3 hypervariable region are listed as successively, the aminoacid sequence of described hypervariable region and EP 449, the Seq.Id.No.1 shown in the 769B1 is identical, EP449, the content of 769B1 is incorporated herein for referencial use at this,

B) comprise second domain of CDR1 ', CDR2 ' and CDR3 ' hypervariable region successively, the aminoacid sequence of hypervariable region and EP 449, the Seq.Id.No.2 shown in the 769B1 is identical, EP449, the content of 769B1 this be incorporated herein for referencial use and

C) with the terminal peptide linker that is connected with the N-end of second domain that be connected or terminal of the terminal C-of the N-of first domain with the C-of first domain with second domain.

According to the present invention, the CD25 binding molecule also can be selected from the strand binding molecule that comprises antigen binding site, and described antigen binding site contains humanized antibody.As daclizumab, at EP 451, this antibody and preparation method thereof is disclosed among the 216B1, EP 451, and the content of 216B1 is incorporated herein for referencial use at this.

As everyone knows, the minor alteration of aminoacid sequence such as defective, interpolation or replace the equipotential form that, a plurality of or several amino acid all can cause forming urporotein, these forms have identical or essentially identical feature, for example antigen-binding with urporotein.Therefore, term " directly equivalent " is meant the CD25 binding molecule (molecule X) in any single structure territory:

(i) wherein said CDR1, CDR2 and CDR3 hypervariable region are done as a whole and EP449, the hypervariable region of Seq.Id.No.1 shown in the 769B1 or EP451, the hypervariable region shown in Fig. 3 and 4 has at least 80% to be homologous among the 216B1, preferably have at least 90% to be homologous, more preferably have at least 95% to be homologous, the content of these patent documentations is incorporated herein for referencial use at this; With

(ii) identical with molecule X but hypervariable region CDR1, CDR2 and CDR3 and EP449 with framework region, Seq.Id.No.1 shown in the 769B1 or EP451, Fig. 3 among the 216B1 compares with the identical contrast molecule shown in 4, this binding molecule is basic identical with the inhibition degree of its receptors bind to interleukin II (IL-2), and the content of these patent documentations is incorporated herein for referencial use at this;

Refer to that perhaps each binding site has any CD25 binding molecule (molecule X ') of at least two domains

(i) wherein said CDR1, CDR2, CDR3, CDR1 ', CDR2 ' and CDR3 ' hypervariable region are done as a whole and EP449, Seq.Id.No.1 shown in the 769B1 and 2 hypervariable region or EP451, the hypervariable region shown in Fig. 3 and 4 has at least 80% to be homologous among the 216B1, it is 90% homologous preferably having at least, and the content of these patent documentations is incorporated herein for referencial use at this;

(ii) identical with molecule X ' but hypervariable region CDR1, CDR2 and CDR3 and EP449 with framework region, Seq.Id.No.1 shown in the 769B1 and 2 or EP451, Fig. 3 among the 216B1 compares with the identical contrast molecule shown in 4, this binding molecule is basic identical to the inhibition degree of IL-2 and its receptors bind, and the content of these patent documentations is incorporated herein for referencial use at this.

Last standard can very convenient check in different tests, as EP449, and 769B1 or EP451, described in the 216B1, the content of these patent documentations is incorporated herein for referencial use at this.According to the present invention, preferred CD25 binding molecule is the antibody of chimeric CD25, particularly comprises the chimeric CD25 antibody of the chain below at least one:

A) comprise the heavy chain of variable domains and people's heavy chain constant portion, the aminoacid sequence of described variable domains and EP449, sequence shown in the Seq.Id.No.1 is identical or basic identical among the 769B1, the content of this patent documentation is incorporated herein for referencial use at this, initial by 1 amino acids, stop by 117 amino acids; With

B) comprise the light chain of variable domains and people's light chain constant portion, the aminoacid sequence of described variable domains and EP449, sequence shown in the Seq.Id.No.2 is identical or basic identical among the 769B1, the content of this patent documentation is incorporated herein for referencial use at this, originate in 1 glutamic acid, end at 104 glutamic acid.

The constant portion of people's heavy chain can be γ ¹, γ ², γ ³, γ ⁴, μ, α ¹, α ², δ or ε type, be preferably the γ type, more preferably γ ¹Type (comprises λ and the constant portion of people's light chain can be κ or λ type ¹, λ ²And λ ³Hypotype), be preferably the κ type.The aminoacid sequence of all these constant portions by Kabat etc. at Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, Public Health Service, NIH provides.

According to the present invention, most preferred CD25 binding molecule is chimeric antibody basiliximab (basiliximab), with SIMULECT ^Trade name derive from Novartis AG and humanized antibody daclizumab, with ZENAPX ^Trade name derive from Roche.

According to the present invention, suitable CD25 binding molecule can be with reference to EP449,769B1 and EP451, and the disclosed production method production of 216B1, the content of these patent documentations is incorporated herein for referencial use at this.

As EP-B-449,769B1 and EP451, described in the 216B1, in measuring, can be observed on the active basis of CD25 binding molecule, have been found that the CD25 binding molecule is applicable to autoimmune hepatitis, asthma, eczema, vasculitis, the property a crossed arthritis, systemic lupus erythematosus (sle), leukemia, glomerulonephritis, multiple sclerosis, ulcerative colitis, sarcoidosis, the segmental enteritis of prevention and treatment steroid responsive type patient or resistant patients as lymphocyte steroid sensitivity (Lymphocyte Steroid Sensitivity).

Therefore, the present invention also provides:

(i) method of the autoimmune hepatitis of prevention and treatment steroid responsive type patient or steroid-resistant patients, asthma, eczema, vasculitis, the property a crossed arthritis, systemic lupus erythematosus (sle), leukemia, glomerulonephritis, multiple sclerosis, ulcerative colitis, sarcoidosis or segmental enteritis, this method comprises the above-mentioned CD25 binding molecule that gives described patient's effective dose.

(ii) prevent and treat the autoimmune hepatitis of steroid responsive type patient or steroid-resistant patients, asthma, eczema, vasculitis, one property crossed arthritis, systemic lupus erythematosus (sle), leukemia, glomerulonephritis, multiple sclerosis, ulcerative colitis, the method of sarcoidosis or segmental enteritis, this method comprises and giving the following material of (for example simultaneously or successively) described patient's effective dose: a) aforesaid CD25 binding molecule, and b) effectively prevent and treat autoimmune hepatitis, asthma, eczema, vasculitis, one property crossed arthritis, systemic lupus erythematosus (sle), leukemia, glomerulonephritis, multiple sclerosis, ulcerative colitis, the other medicine of sarcoidosis or segmental enteritis.

(iii) be used for (i) and reach the (ii) pharmaceutical composition of described method, said composition comprises aforesaid CD25 binding molecule and pharmaceutically acceptable carrier or diluent.

(iv) be used for producing the aforesaid CD25 binding molecule of the medicine that (i) or (ii) described method use.

(v) be used for (i) or the (ii) therapeutic combination of described method, as medicine box or pharmaceutical pack, described combination comprises the pharmaceutical composition of the CD25 of comprising binding molecule recited above, and also comprise the pharmaceutical composition of the medicine of at least a another treatment steroid responsive type patient or the following disease of resistant patients that comprises effective dose: autoimmune hepatitis, asthma, eczema, vasculitis, are crossed property arthritis, systemic lupus erythematosus (sle), leukemia, glomerulonephritis, multiple sclerosis, ulcerative colitis, sarcoidosis or segmental enteritis.

When used according to the invention, suitable dosage should change according to following factor certainly, as used concrete CD25 binding molecule, patient's s own situation, administering mode, the order of severity of the state of an illness and required effect.Drug dose from about 0.1mg to about 100mg all can obtain satisfied curative effect usually.Can single dose administration, also can in a period of time, divide a plurality of dosed administrations, as long as obviously or can the prevent disease recurrence at clinical effectiveness, for example about 5mg is to the dosage of about 100mg, can administration in a day to five weeks, as the controlled or improvement that was administered once in per 3 to 6 days up to disease.Preferred dosage regimen was, gave CD25 binding molecule 20mg (as basiliximab) at the 0th day, gave 20mg on the 4th day again.Preferably,, for example pass through before the elbow or other peripheral vein blood vessel administration as intravenously administrable with CD25 binding molecule parenteral.An exemplary dosage regimen in addition is per 28 days intravenously administrable 40mg, the controlled or improvement up to disease.

Venoclysis is penetrated liquid and can be prepared as follows: freeze dried antibody is mixed and be scattered in the aseptic buffer salt solution of 100ml, it contains weight is 4.5% human albumin.Can give the patient by vein heavy dose (bolus) injection or at 15 minutes angular vein infusions with this salt dispersion liquid.

Studies show that up to the present, giving the CD25 binding molecule in described dosage range does not have unacceptable side-effects.Particularly, preferred medicine basiliximab or daclizumab are safe, by drug administration of the United States Federal (FDA) approval, can commercially use.

According to EP449,769B1 or EP451, the method described in the 216B1 can prepare pharmaceutical composition of the present invention, and the content of these patent documentations is incorporated herein for referencial use at this.

The CD25 binding molecule can be used as independent active component or together uses with other immunomodulating or anti-inflammatory drug.For example, according to the present invention, the CD25 binding molecule can with the active drug compositions drug combination of the aforesaid various diseases of various treatments, for example cyclosporin, rapamycin or ascosin or their inhibitory analogues are as cyclosporin A, cyclosporin G, FK-506 (Prograf), sirolimus, everolimus; The glucocorticoid class is as prednisone; Cyclophosphamide; Azathioprine; Methotrexate; Gold salt; Sulfasalazine, antimalarial agent, Boulez Kui sodium; Leflunomide; Mizoribine; Mycophenolic acid; Mycophenolic acid is ester (mycophenolate mofetil) not; The smart melon element (15-deoxyspergualine) of 15-deoxidation; Other inhibitive ability of immunity monoclonal antibodies are as monoclonal antibody such as MHC, CD2, CD3, CD4, CD7, CD28, B7, CD40, CD45 or CD58 or their part of anti-leukocyte receptors; Perhaps other immunomodulatory compounds such as CTLA41g.

If with CD25 binding molecule and other medication combined medication, two kinds of medicines can be packed separately, place then in the same container, and put into the description about mixing or administration simultaneously in container.The example of medicine box comprises as injection syringe with multi-cavity chamber or comprises the independently double pack of dosage form.

Concrete embodiment

The following examples are used to illustrate the present invention.

Embodiment 1

The object of study of this experiment is 4 man and 4 woman.The mean age of object of study is 52 years old (from 34 years old to 70 years old), and wherein two object of study are healthy volunteers, and other six object of study are the patients that are diagnosed as ulcerative colitis early stage.Wherein 5 patients had before required operative treatment because of medical treatment is invalid, existing any antiinflammatory or the immunosuppressant do not taken.The 6th object of study is UC (ulcerative colitis) patient's resting stage, and he has taken 5-aminosalicylic acid, though be not to take a sample the same day, therefore, had the 5-aminosalicylic acid metabolism of inhibition interleukin II effect during sampling.(the commercialization SIMLECT by name that is used for external basiliximab ^, provide by Novartis)

Measure lymphocyte to steroid sensitivity: peripheral blood lymphocyte can be measured in function test the sensitivity of glucocorticoid, the external hypertrophy test that just lectins (PHA) stimulates, dexamethasone suppresses (Hearing SD etc., Gut 1999; 45:382-8; Hearing SD etc., J Clin Endocrinol Metab 1999; 84:4149-54).Peripheral blood sample is gathered at 9:00, with buoyancy centrifuging separating periphery blood monocytic cell (PBMC).Buoyancy is centrifugal to be comprised by Ficol gradient centrifugation isolated lymphocytes from blood sample.Ficol filters erythrocyte, and stops lymphocyte to pass through.Collect peripheral blood lymphocytes (lymphocyte and megalocyte) at the interface from ficol/ blood plasma.10 ^-6-10 ^-10Mol/l is 10 ^-6, 10 ^-7, 10 ^-8, 10 ^-9With 10 ^-10With the diluted lectins and the dexamethasone of continuous 10 multiples, the former stimulates peripheral blood lymphocytes between the mol/l, and the latter suppresses peripheral blood lymphocytes.The absorbtivity of the thymidine by measuring tritiate is measured hyperplasia, just with the thymidine of labelled with radioisotope ( ³The H-thymidine) add in the lymphocyte culture fluid, through 2 days incubation, it mixed among the lymphocytic DNA of hypertrophy.After 6 hours, cell is shifted out incubator, be collected on the filter pad with cellular collector.Then these cells are washed out from the hole.The lymphocyte DNA that mixes thymidine remains on the filter paper.When measuring radioactivity, have in the hole of the outgrowth lymphocyte of a large amount of activity (and therefore having a large amount of thymidines to mix among the DNA) very high radioactivity will be arranged.Therefore, with radioactive level (CPM represents with per minute radiation number) as lymphoproliferative index.The result calculates with the average count per minute (CPM) of three parts of cultures.The lymphocytic number that mixes of T that the thymidine of the dexamethasone of each concentration is mixed lectins that number and no dexamethasone suppress and stimulates is compared, and represents with the percent of the positive control of inhibition not.The basiliximab of also having studied variable concentrations is to lymphoproliferative influence.In addition, in each cell culture fluid of the dexamethasone that contains variable concentrations, all add the basiliximab of same concentrations, measure the effect of its inhibition of cell proliferation.

SIMULECT ^The basiliximab serum-concentration of lymphoproliferative effect: 0.2mg/l has been occupied interleukin II receptor more than 90%.In order to measure the external anti-hypertrophy effect of basiliximab, be that the basiliximab of the variable concentrations of 0.01-10mg/l is cultivated in the presence of no steroid with lymphocyte with concentration range.These test confirmation, and basiliximab is 0.1-0.3mg/l in the concentration of external generation effect.In the other research that these objects are carried out, adopt the concentration of 1mg/l.Have in a word, in each test, all studied and add up to 8 same object.Under the situation that no steroid exists, concentration is that the basiliximab of 1mg/l acts on that the lectins inducing cell is outgrowth to be the results are shown in the table 1:

Table 1

Object of study	1	2	3	4	5	6	7	8	On average
										Independent lectins (cpm)	38634	25228	40829	32561	45441	66157	28886	36990	39341
Lectins and basiliximab (cpm)	21827	15157	27239	27538	21446	35003	16122	27209	23943
										Suppress percentage rate	43.5	39.9	33.3	15.4	52.8	47.1	44.2	26.4	37.8

Therefore under the situation that no steroid exists, add the hypertrophy inhibition that basiliximab has produced an average out to 37.8%.Yet, this inhibition level still relevant (＜60%) with the clinical efficacy difference of patients of ulcerative colitis in the research.

SIMULECT ^Effect to steroid sensitivity: increase progressively (10 at dexamethasone concentration ^-10-10 ^-6Mol/l) lymphocyte that in the culture fluid lectins is stimulated is cultivated, and will be divided into two groups to 8 object of study, adds basiliximab (1mg/l) group and does not add the basiliximab group.Formerly in the research of the ulcerative colitis that the inventor did, maximum suppresses (I _MaxThe curative effect of)＜60% o'clock steroid relatively poor (clinical steroid toleration).I _MaxExplicit value be unmeasured because the inhibitory action that it is produced when being dexamethasone infinite height level.Therefore, will be at used dexamethasone Cmax (10 ^-6Mol/l) percent that the hypertrophy of measuring the time suppresses is as I _MaxValue.In the practical application, the maximum that is estimated to by beneficial effect curve suppresses asymptotic value and I _Max((1999) Clin.Endoctrinol.Metab 84:4149-54 such as Hearing SD) well conforms to.With the activity of glucocorticoid, 10 ^-7The dexamethasone concentration of mol/l is about as much as the concentration of the hydrocortisone that intravenous injection is reached under the standard drug dosage in serum.Therefore, in order to compare basiliximab potential effect in vivo, people are also very interested in the steroid sensitization of basiliximab the dexamethasone under this concentration.Table 2 has shown the result of each object of study in the lymphocytosis experiment, and this result shows the effect of basiliximab in lymphocyte steroid sensitivity.The sequence arrangement that object of study is successively decreased with steroid sensitivity (left side is for the most responsive).

Table 2

Independent lectins (cpm)	38634	25229	40829	32562	45442	66157	28887	36991
									Lectins and 10 -6Dexamethasone (cpm)	3100	4109	9477	11568	21685	36495	18669	24681
Lectins and basiliximab and 10 -6Dexamethasone (cpm)	639	4533	3059	3801	3563	6910	3352	5924
									I max(dexamethasone) (%)	92	84	77	64	52	45	35	33
I max(dexamethasone and basiliximab) (%)	98	82	93	88	92	90	88	84
									10 -7Dexamethasone suppresses %	87	79	65	42	20	32	29	36
10 -7Dexamethasone and basiliximab suppress %	98	84	88	83	87	83	86	81
									Steroid responsive type (SS) or steroid tolerance type (SR)	SS	SS	SS	SS	SR	SR	SR	SR

4 object of study are steroid responsive type (maximum inhibition＞60%, 1-4 name), and 4 is steroid tolerance type (maximum inhibition＜60%, mat woven of fine bamboo strips 5-8 name).From the maximum suppression effect of all steroid tolerance type object of study, basiliximab can be regulated the sensitivity of lymphocyte to steroid, all steroid responsive types that all become.And be 10 in concentration ^-7In the dexamethasone of mol/l, also can see the effect that lymphocyte is suppressed percentage ratio.

Basiliximab is regulated lymphocyte steroid sensitivity, equally also can be used for changing the steroid sensitivity of the steroid-resistant patients of following disease: autoimmunity active hepatitis, asthma, eczema, vasculitis, are crossed property arthritis, systemic lupus erythematosus (sle), leukemia, glomerulonephritis, multiple sclerosis, ulcerative colitis, sarcoidosis or segmental enteritis, and invalid being likely because of it of above-mentioned disease patient's steroid therapy has steroid tolerance type lymphocyte.Basiliximab makes steroid tolerance type lymphocyte become the steroid sensitivity cell to the adjusting of lymphocyte steroid sensitivity, and this adjusting makes the patient by before invalid effective to steroid therapy to steroid therapy.In addition, those are considered to the effective steroid responsive type of steroid therapy patient, if use the basiliximab treatment, the onset of steroid therapy is faster with making.

Embodiment 2

Use the buoyant density centrifugation method, divide 41 times and from 32 object of study (25 healthy volunteers, 7 resting stage patients of ulcerative colitis), separate peripheral blood lymphocyte.Under the lymphoproliferative situation that lectins stimulates,, measure the sensitivity of lymphocyte to steroid by measuring the anti-proliferative effect fruit of dexamethasone (DEX).The inductive maximum inhibitory action of steroid is represented (I with the lymphoproliferative percentage ratio of matched group _Max).Having and not having in two groups that basiliximab (1mg/l) exists, measure I respectively _MaxAdd basiliximab and obviously strengthened a dexamethasone anti-proliferative effect for.

(Wilcoxon signed-rank check, p＜0.0001)

	I maxAverage (%)	The spacing of quartile
			Dexamethasone	78.0	66.1-86.4
Dexamethasone and basiliximab	92.3	89.3-95.6

All be adjusted to steroid responsive type (Imax＜60%) by adding all 8 steroid tolerance type object of study (external) of basiliximab.Therefore, basiliximab has a tangible steroid reinforced effects external.

Sequence table

＜110〉Univ. of Bristol (University of Bristol)

＜120〉purposes of CD25 binding molecule in steroid-resistant patients

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Claims (8)

1. the material that is selected from basiliximab and daclizumab is used for preventing or treating steroid responsive type patient autoimmune hepatitis, asthma, eczema, vasculitis, a purposes of the medicine of property arthritis, systemic lupus erythematosus (sle), leukemia, glomerulonephritis, multiple sclerosis, ulcerative colitis, sarcoidosis or segmental enteritis excessively in preparation.

2. according to the purposes of claim 1, wherein said material is a basiliximab.

3. according to the purposes of claim 1, wherein said material is a daclizumab.

4. according to the purposes of claim 1, wherein said material and the steroid administering drug combinations that is selected from basiliximab and daclizumab.

5. according to the purposes of claim 1, the medication combined administration of following disease is effectively treated or prevented to the wherein said material that is selected from basiliximab and daclizumab and another: autoimmune hepatitis, asthma, eczema, vasculitis, are crossed property arthritis, systemic lupus erythematosus (sle), leukemia, glomerulonephritis, multiple sclerosis, ulcerative colitis, sarcoidosis or segmental enteritis.

6. according to the purposes of claim 5, wherein said other medicine is at least a of following medicine: cyclosporin, rapamycin, ascosin, glucocorticoid class, cyclophosphamide, azathioprine, methotrexate, golden salt, sulfasalazine, antimalarial agent, Boulez Kui sodium, leflunomide, mizoribine, mycophenolic acid, mycophenolic acid be ester, the smart melon element of 15-deoxidation, inhibitive ability of immunity monoclonal antibody or its part and immunomodulatory compounds not.

7. according to the purposes of claim 6, at least a administering drug combinations of wherein said CD25 binding molecule and following medicine: cyclosporin A, cyclosporin G, sirolimus, everolimus, prednisone, FK-506, rapamycin, mycophenolic acid, mycophenolic acid be monoclonal antibody or its part and the CTLA41g of ester, the smart melon element of 15-deoxidation, anti-leukocyte receptors not.

8. according to the purposes of claim 7, wherein said monoclonal antibody is the monoclonal antibody of CD28, CD40 or its mixture.