CN101022828A - Inhibition of factor b, the alternative complement pathway and methods related thereto - Google Patents

Abstract

Disclosed are novel inhibitors of the alternative complement pathway and particularly, novel anti-factor B antibodies. Also disclosed is the use of such inhibitors to reduce or prevent airway hyperresponsiveness and/or airway inflammation by selectively inhibiting the alternative complement pathway, thereby treating diseases in which such conditions play a role. Also disclosed is the use of such inhibitors to reduce or prevent other diseases and conditions, including ischemia-reperfusion injury, by inhibition of the alternative complement pathway.

Description

The inhibition of the B factor, complement bypass and relevant therewith method

Technical field

The present invention relates generally to the new inhibitor of complement bypass, particularly novel anti-factor B antibodies.The present invention relates to also generally that this inhibitor reduces or prevention airway hyperreactivity and airway inflammation, treat the purposes of the disease that this disease works thus.

Background technology

Complement activation is mainly by three kinds of approach: so-called classical pathway, lectin pathway and alternative pathway.Participating in the activated key protein of alternative pathway is the B factor (fB) and the D factor (fD).These albumen concur, and to start and/or to amplify the activation of C3, this causes the startup of many inflammatory events then.The third albumen properdin can be stablized the complex of the C3 and the B factor, and is necessary but it is not that alternative pathway works.The B factor also helps the lytic immunity complex, and the effect of the Bcell growth factor that has been in the news and energy activated mononuclear cell (Takahashi, 1980; Hall, 1982; Peters, 1988).Cultivated the mice (fB-/-mice) of B factor disappearance, and in these mices, at the antigenic IgG1 antibody response that relies on the T cell with to the sensitivity of endotoxin shock seemingly normal (Matsumoto, 1997).

Complement bypass is started by antibacterial, parasite, virus or fungus usually, but reports that also IgA Ab and some Ig L chain can activate this approach.As the circulation B factor and activated C3 (C3b or C3H2O) when combining, alternative pathway activates and is activated.This complex is recycled the D factor and shears the fragment C3Bb that generation has the enzyme activity then.C3Bb shears C3 and forms C3b, and this drives inflammation, also further amplifies activation process simultaneously, forms positive feedback loop.Need two kinds of components (the B factor and the D factor) could activate alternative pathway.

The nearest complement bypass that studies show that plays an important role in the pathogeny of several animal disease models.Complement activation behind the I/R in the kidney substantially only is subjected to the mediation (Thurman) of alternative pathway, and alternative pathway plays pivotal role in arthritic progress simultaneously.Especially it is shocking, shown that the mice that lacks alternative pathway can not suffer from nephritis (Watanabe) in lupus nephritis MRL/Ipr model, and can not cause the fetal loss (Girardi) of anti-phospholipid mediation, this pattern is considered to be subjected to the CCP mediation usually.

Developed and severally suppressed the inhibitor (Holers) of complement systems at different active periods, but before the present invention the special inhibitor of alternative pathway wide coverage not as yet.The polypeptide that July 4 calendar year 2001, disclosed PCT publication WO 01/47963 described from ectoparasite Hirudo, it is at the alternative pathway of vitro inhibition complement activation, and it does not have influence substantially to the complement activation by classical pathway.Shown these peptides in conjunction with the D factor, still, undeclared these polypeptide are used in vivo.The reagent of specificity inhibition alternative pathway is compared with the inhibitor of existing complement connection order reaction and is had several respects advantage in theory in vivo.At first, with regard to the model of alternative pathway mediation, as the fetal loss of kidney I/R and the mediation of anti-phospholipid, this inhibitor should be the same with the wide spectrum complement inhibitor effective, also should have less immunosuppressant side effect with regard to basic.Although report has only the human patients (Densen) of a routine congenital B factor disappearance, the research of the B factor disappearance mice of gene targeting (fB-/-) is not found that as yet this factor has immunoregulation effect (Densen; Matsumoto).On the contrary, as if the patient of ectrogeny classical pathway component has bigger infection risk (modal is staphylococcus and streptococcus).Suppressing classical pathway component or C3 (all complement pathways are common) may also relevant with autoimmune (Figueroa), this possible explanation why B factor disappearance make the MRL/Ipr mice can not suffer from glomerulonephritis, and disappearance C3 opposite (Watanabe).Therefore, the inhibition of alternative pathway may have better toleration, and it is more effective than the complement inhibition of classical pathway in some cases.

Allergic asthma is a kind of common airway inflammation and airway hyperreactivity (AHR) related syndromes (Busse).The patient who suffers from allergic asthma causes AHR and airway inflammation to strengthen after being exposed to the anaphylactogen of suction, studies show that fragment level raising, the particularly level of C3a (Humbles) in bronchoalveolar lavage (BAL) liquid and C5a (Krug) raising from the biologically active of complement C3, C4 and C5 protein family.This activation that shows these patients complement pathway by allergen-induced mechanism after being exposed to anaphylactogen appears at pulmonary.Animal model further provides the effect of complement in the allergic airway diseases progress.As if the animal of C3 or C3a receptor disappearance airway disorders (Humbles, the Drouin by allergen-induced can not occur; Bautsch; Walters).

Supposition induces complement activation that several probabilities are arranged after being exposed to anaphylactogen.For example, anaphylactogen-IgG immune complex can excite the activation of classical pathway, and some antigen can directly activate C3 (Kohl) by alternative pathway.In addition, but neutral trypsinlike enzyme direct shearing (Proteolytic enzyme) C3 or C5 (Schwartz that mastocyte or pulmonary macrophage discharge; Mulligan).Three kinds of approach (classical pathway, alternative pathway and lectin pathway) of complement activation meet at center complement component C3.Therefore, the activated inhibition of C3 can prevent that it is sheared the active C3 fragment of formation, has also significantly reduced the downstream activation of C5 and the release (Sahu) that derives from the active fragment of C5.Nearest studies show that when being exposed to anaphylactogen by the sensitization animal suppresses complement activation (suppressing whole three kinds of activated pathway thus) by use C3 converting enzyme inhibitor, weakened the progress (Taube) of air flue reaction in late period (Abe) and slowed down AHR and airway inflammation.The PCT publication WO 2004/022096 that announced on March 18th, 2004 has described the inhibition of complement bypass, the preferred terminal complement component by the total C5-C9 of all approach, the most preferably inhibition by C5a.

At present, the treatment Therapeutic Method that relates to the inflammation disease (as moderate to severe asthma and chronic obstructive disease of lung) of AHR mainly comprises the use of glucocorticoid and other antibiotic medicine.But these medicaments may have serious adverse, include but not limited to improve susceptibility, hepatotoxicity, drug-induced pulmonary disease and bone marrow depression to infecting.Therefore, these medicaments have limitation in the clinical use of the relevant pulmonary disease of treatment airway hyperreactivity.The use of antibiotic medicine and remission medicament is a serious problem, and this is the side effect owing to them, and perhaps they can not attack the basic reason of inflammatory reaction.All need to treat the littler and more efficiently medicament of toxicity of inflammation always.Therefore, need to use have less side effect, toxicity is little and to the more special compositions and methods of basic reason of allergic airway diseases such as asthma and AHR disease.

Summary of the invention

One embodiment of the invention relate to separation antibody or its Fab, and (short consensus repeat, SCR) domain, wherein said antibody have stoped the formation of C3bBb complex to the 3rd short consensus repeat of its selective binding B factor.On the one hand, antibody or its Fab be in conjunction with the B factor, and prevent or suppress the D factor and shear the B factor.On the other hand, antibody or Fab are in conjunction with the 3rd short consensus repeat (SCR) domain of the people B factor.On the other hand, antibody or Fab combination are selected from the epi-position of the Three S's CR domain of the following B factor: the epi-position that (a) comprises the B factor of at least a portion people B factor (SEQ IDNO:2), comprise from about Tyr139 position to about Ser185 position, or its equivalent position in non-human B factor sequence; (b) comprise the epi-position of the B factor of at least a portion people B factor (SEQ ID NO:2), comprise from about Try139 position to about Ser141 position, or its equivalent position in non-human B factor sequence; (c) comprise the epi-position of the B factor of at least a portion people B factor (SEQ ID NO:2), comprise from about Glu182 position to about Ser185 position, or it is at the equivalent position of non-human B factor sequence; Perhaps (d) comprises the epi-position of the B factor of at least a portion people B factor (SEQ ID NO:2), comprise any one or a plurality of down column positions or its equivalent position in non-human B factor sequence: Tyr139, Cys140, Ser141, Glu182, Gly184 or Ser185.On the other hand, the epi-position of the Three S's CR domain of antibody or its Fab selective binding B factor (SEQ ID NO:2), this epi-position comprises one or more following amino acid positions or its equivalent position in non-human B factor sequence: Ala137, Tyr139, Ser141, Glu182, Ser185, Thr189, Glu190 and Ser192.On the other hand, the epi-position of the Three S's CR domain of antibody or its Fab selective binding B factor (SEQ ID NO:2), this epi-position comprises following amino acid position or its equivalent position at non-human B factor sequence: Ala137, Tyr139, Ser141, Glu182, Ser185, Thr189, Glu190 and Ser192.On the other hand, the epi-position of the Three S's CR domain of antibody or its Fab selective binding B factor (SEQ ID NO:2), this epi-position is made up of following amino acid position or its equivalent position at non-human B factor sequence: Ala137, Tyr139, Ser141, Glu182, Ser185, Thr189, Glu190 and Ser192.This antibody or Fab can be in conjunction with the non-linear epi-positions in the three dimensional structure of the part of the Three S's CR domain of the B factor, wherein this part is defined as being at least the amino acid position Ala137-Ser192 of SEQ ID NO:2, or its equivalent position in non-human B factor sequence.On the other hand, the B factor of antibody or the multiple mammalian species of its Fab selective binding (as the mankind and the animal that is selected from primates, mice, rat, pig, horse and rabbit except that the mankind).Antibody or Fab can be non-complement activation isotype or subclass, can be monoclonal antibody, humanized antibody, bi-specific antibody or univalent antibody.Fab can comprise the Fab fragment.In a preferred embodiment, antibody is monoclonal antibody 1379 (being produced preserving number PTA-6230 by ATCC).

Another embodiment of the invention relates to separation antibody or its Fab of the B factor of the multiple mammalian species of selective binding, and wherein this antibody has stoped the formation of C3bBb complex.On the one hand, antibody or its Fab selective binding people and the B factor that is selected from the animal of primates, mice, rat, pig, horse and rabbit except that the people.On the one hand, antibody is non-complement activation isotype or subclass.On the other hand, antibody is monoclonal antibody.On the other hand, Fab is the Fab fragment.

Another embodiment of the present invention relates to antigen-binding polypeptides, the 3rd short consensus repeat (SCR) domain of its selective binding B factor, wherein said antigen-binding polypeptides has stoped the formation of C3bBb complex, the antigen-binding polypeptides that perhaps relates to the B factor of the multiple mammalian species of selective binding, wherein this antigen-binding polypeptides has stoped the formation of C3bBb complex.

Another embodiment of the present invention relates to separation antibody or its Fab of the selective binding B factor, wherein said antibody or its fragment competitive inhibition monoclonal antibody 1379 (are produced by ATCC, preserving number PTA-6230) to the specificity combination of the people B factor, and wherein, when antibody or its Fab during in conjunction with the people B factor, the ability that monoclonal antibody 1379 suppresses complement bypasses is suppressed.On the one hand, antibody or its Fab competitive inhibition monoclonal antibody 1379 are in conjunction with the people B factor, and when the people B factor exist detect relatively binding specificity by antibody-antibody competition analysis this moment.

Another embodiment of the present invention relates to separation antibody or its fragment of the selective binding people B factor, anti-or its Fab specificity of wherein said separation antibody or its fragment competitive inhibition two is in conjunction with the people B factor, and wherein said two anti-or its Fabs are in conjunction with the Three S's CR domain of the people B factor.

The present invention also comprises the compositions that comprises any above-mentioned antibody, Fab or antigen-binding polypeptides.

Another embodiment of the present invention relates to reduction or the airway hyperreactivity (AHR) of prevention animal or the method for airway inflammation.This method comprises the steps: that above-mentioned antibody or its Fab are suffered from inflammation relevant airway hyperreactivity or airway inflammation or the animal that suffers from this disease risk is arranged.On the one hand, give antibody or Fab by following approach: in oral, nose, part, suction, the trachea, percutaneous, rectum or parenteral route.On the other hand, with antibody or Fab with the amount that effectively significantly reduces the airway hyperreactivity of animal (with give antibody or Fab before compare) give animal.On the other hand, give animal with antibody or Fab with the amount that effectively significantly reduces the airway hyperreactivity of animal (compare with the level of the airway hyperreactivities of many animals that inflammation arranged, wherein do not give described antibody or Fab).On the one hand, antibody or Fab has reduced the reactivity of animal to acetonyl choline or histamine.On the other hand, antibody or Fab are selected from following carrier and give with pharmaceutically useful: can disperse dry powder, dehydrated alcohol, Caplet, liposome, atomisation agent and injectable excipient.On the other hand, antibody or Fab can give in being selected from following carrier or device: dehydrated alcohol, dry powder intake system, ultrasonic intake system, pressure measurement inhaler and metering solution device.On the other hand, antibody or Fab give described mammal with being selected from following reagent: the inhibitor of corticosteroid, beta-2-agonists (long-acting or fugitive), leukotriene instrumentality, antihistamine, phosphodiesterase inhibitor, sodium cromoglicate, nedocromil (Nedocromil), theophylline, cytokine antagonist, cytokine receptor antagonist, anti-IgE and T cell function.On the other hand, airway hyperreactivity or airway inflammation and be selected from following disease association: asthma, chronic obstructive disease of lung (COPD), anaphylaxis broncho-pulmonary aspergillosis, hypersensitivity pneumonitis, the eosinophilic granulocyte pneumonia, emphysema, bronchitis, allergic bronchitis, bronchiectasis, cystic fibrosis, tuberculosis, hypersensitivity pneumonitis, occupational asthma, sarcoidosis, RADS, interstitial lung disease, hypereosinophilic syndrome, rhinitis, sinusitis, exercise-induced asthma, asthma (pollution-induced asthma) is brought out in pollution, cough variant asthma, parasitic disease of lung, respiratory syncytial virus (RSV) infects, parainfluenza virus (PIV) infects, rhinovirus (RV) infects and adenovirus infection.On the one hand, airway hyperreactivity is relevant with allergic inflammation.In a preferred embodiment, method of the present invention can be implemented in mammal, more preferably implements in the people.

Description of drawings

Fig. 1 illustrates the sketch map that makes up the B factor-Ig fusion rotein.

Fig. 2 A illustrates the anti-B factor of 3 μ g to add when containing the reaction system of 10 μ l serum, and the anti-B factor suppresses the line chart of complement bypass fully in the zymosan analysis.

When Fig. 2 B added 10 μ l human serums for the anti-B factor antibody of 6 μ g is shown, this anti-B factor had suppressed the line chart of complement bypass fully in the rabbit reticulocyte lysate analysis.

Fig. 3 illustrates to give the line chart that mice suppresses complement bypass with the anti-B factor.

Fig. 4 A is airway resistance (R _L) line chart, this illustrate the fB+ of anaphylactogen sensitization and attack /+mice and the fB+ that is only attacked /+mice compares its reactivity to the acetonyl choline and strengthens, but fB-/-mice significantly reduces the reactive of acetonyl choline.

Fig. 4 B is the line chart of Cdgn dyanamic compliance (Cdyn), this illustrate the fB+ of anaphylactogen sensitization and attack /+mice and the fB+ that is only attacked /+mice compares its reactivity to the acetonyl choline and strengthens, but fB-/-mice significantly reduces the reactive of acetonyl choline.

Fig. 5 for characterize the air flue sensitization and attack after fB-/-the BAL liquid of mice and the block diagram of lung tissue.

Fig. 6 A is airway resistance (R _L) line chart, this illustrate the fB-of artemisiifolia sensitization and attack/-mice reduces the reactivity of acetonyl choline, but fB+ /+mice is stronger to the reactivity of acetonyl choline.

Fig. 6 B is the line chart of Cdgn dyanamic compliance (Cdyn), this illustrate the fB-of artemisiifolia sensitization and attack/-mice reduces the reactivity of acetonyl choline, but fB+ /+mice is stronger to the reactivity of acetonyl choline.

Fig. 6 C is for characterizing the block diagram of BAL liquid and lung tissue, this illustrate the fB-of artemisiifolia sensitization and attack/-airway inflammation in the BAL liquid of mice and fB+ /+mice compares and decreases.

Fig. 7 A is a line chart, this illustrate before each the attack with the B factor handle by the fB-of sensitization and attack/-mice is to the reactivity reduction of acetonyl choline, this to handle with PBS by the fB-of sensitization and attack/-mice is similar, but with by the fB+ of sensitization and attack /+mice compares remarkable reduction.

Fig. 7 B be illustrate the B factor give fB-/-mice in reconstruct the block diagram of the ability of AHR and airway inflammation appears.

Fig. 8 A is airway resistance (R _L) line chart, this illustrates whole body and spraying gives B factor neutralizing antibody and suppressed by the progress of AHR in the mice of sensitization and attack.

Fig. 8 B is the line chart of Cdgn dyanamic compliance (Cdyn), and this illustrates whole body and spraying gives B factor neutralizing antibody and suppressed by the progress of AHR in the mice of sensitization and attack.

Fig. 8 C is for characterizing the block diagram of BAL liquid and lung tissue, this illustrates with anti-B factor whole body or spraying processing and has reduced the quantity of the eosinophilic granulocyte in the BAL liquid, peribronchitis disease, bronchus week eosinophilic granulocyte's quantity and the quantity of the mucus positive cell in the airway epithelia.

Fig. 9 is a line chart, this illustrate by the C4-of sensitization and attack/-mice (solid diamond, n=10) to the MCh that sucks show with by the C4+ of sensitization and attack /+mice (closed square, n=10) similar reactivity, and with the C4-that is only attacked/-mice (open diamonds, n=10) and the C4+ that is only attacked /+mice (hollow square, n=10) compare and reactive significantly strengthen (* with by the fB-of sensitization and attack/-mice, attacked fB+ /+mice and the fB-that is attacked/-mice compares p＜0.05; # with by the fB+ that attacked /+mice and the fB-that is attacked/-mice compares p＜0.05;

With by the C4+ that attacked /+mice and the C4-that is attacked/-mice compares p＜0.05).

Figure 10 is a line chart, this illustrate by the C4-of sensitization and attack/-mice (solid frame, n=8) with the C4-that only attacked/-mice (hollow frame, n=8) comparing its airway resistance to the MCh of suction strengthens, it also shows with anti-B factor monoclonal antibodies whole body handle by the C4-of sensitization and attack/-mice reduced its airway reactivity to MCh (solid circles, n=8).

Figure 11 is a sketch map, and this illustrates the epi-position graph model at the lip-deep mAb1379 of the people B factor.

Figure 12 is a sketch map, and this illustrates the modelling complex of mAB1379 (a Fab fragment) in conjunction with the B factor, and the antigen binding end of Fab is by the model of the epitope regions of all drawing as covering.

Figure 13 is a block diagram, and this illustrates, and to compare the level that serum urea nitrogen raises after pouring into 24 hours again with the contrast of 1379 pretreated mices and wild type lower.

The specific embodiment

One embodiment of the invention relate to provides the novel B of selective exclusion complement bypass factor antibody, and relates to this antibody and be suppressed at that to need this inhibition, this inhibition be useful or by the useful any disease of expectation or the purposes of the complement bypass in the disease.Particularly, consider inhibitor the preferably potential therapeutic effect during being used for the treatment of the method for multiple disease of specificity at complement bypass, the inventor has developed several novel inhibition type monoclonal antibodies at the B factor.Identified several antibody, and wherein a kind of antibody is identified very at length.This antibody is at allergic inflammation of accepting extensively and asthmatic model and check in vitro and in vivo in kidney ischemia reperfusion injury model (being applied to ischemia reperfusion injury usually).For producing this antibody, with the B factor disappearance mice of fusion rotein injection gene targeting (fB-/-), this fusion rotein is made up of the second and the 3rd short consensus repeat (SCR) domain of the B factor that links to each other with immunoglobulin.Just to the immunne response of B factor screening mice, and will be fused to the myeloma cell from the splenocyte of an injection mice wherein.Resulting wherein a kind of hybridoma-being called 1379-production suppresses the activated IgG of complement bypass in vitro and in vivo ₁Antibody has the multiple monoclonal antibody (referring to table 4) that suppresses the complement bypass ability although the inventor has produced and identified.1379 antibody (being also referred to as mAb1379 in this article) suppress to activate from the alternative pathway in the serum of multiple animal species, and these animals comprise mice, rat, people, baboon, macaque, stump-tailed macaque (cyno monkey), pig, rabbit and horse.Fab fragment from this antibody also can suppress alternative pathway fully.The inventor shows that also this antibody can suppress human serum fully to erythrocytic cracking, and definite thus this reagent is blocked the activated ability of complement bypass fully.Epi-position figure is used to illustrate the 3rd short consensus repeat (SCR) domain of this antibodies B factor, and this antibody has stoped the formation of C3bBb complex.Vide infra about detailed description by the epi-position of this antibody recognition.Therefore, one embodiment of the invention relate to the selective depressant of complement bypass, be particularly related to those novel B factor antibody, they have the species reactivity of broad, proved effectiveness in vitro and in vivo, and be to be used for various disease conditions and the effective treatment tool of any camber of disease, the selectivity inhibition to complement bypass in these diseases is useful, necessary and/or preferred (as the disease (vide infra) relevant with airway hyperreactivity and airway inflammation, ischemia reperfusion injury or the like).These antibody also can or carry out other processing to reduce from immune potential side effect by humanization, become a kind of valuable novel therapeutic medicament thus.

Site on the common B factor of several mammals of the antibody recognition that the present inventor produced (comprising the people), in these several mammals, carry out preclinical Proof-Of Principle experiment, therefore made the discovery in the human diseases model be easy to be transformed into the human treatment.Before the present invention, any other that the inventor does not know at the B factor shows the antibody that this albumen is had broad species inhibition as antibody of the present invention.Therefore, the present invention has also identified site unique on the B factor, but at this site development of new inhibitor.The B factor and other proteic evaluation as particular treatment target material in the complement bypass provide reasonable treatment and can treat the inflammatory diseases of air flue and the main compound of other disease.The selective exclusion alternative pathway has some advantages.For example, C4-/-mice (lacking the common C4 complement component of classical pathway, alternative pathway and agglutinin complement pathway) rather than fB-/-as if (B factor disappearance) mice more responsive to the experiment bacterial infection, this shows that the inhibitor of alternative pathway has than low-risk severe infections by not destroying classical pathway.The blocking-up of classical pathway also can cause autoimmune, and the patient of congenital classical pathway component disappearance is infected and the autoimmune risk of generation increases.Selectivity suppresses the feasible part that derives from C3 that can not form in conjunction with C3a receptor and conjugated complement receptor 1-4 and C5a of alternative pathway.Because it is indeterminate that the Ba of the B factor that forms in activation process or Bb activate the receptor sign of product, the influence of blocking-up alternative pathway in fact may be more direct.

Another embodiment of the present invention relates to the inventor's uncommon discovery, and this discovery shows that be vital by alternative pathway activating complement connection order reaction in the progress of airway hyperreactivity and airway inflammation, and is actually necessary and sufficient.More specifically, the inventor discloses alternative pathway but not the inhibition of CCP prevention airway hyperreactivity and reduce the discovery of airway inflammation in this article.The inventor uses the mice (promptly by the gene knockout technology) of B factor disappearance and confirms this discovery by the use monoclonal antibody inhibition B factor (send pass by whole body or aerosol).Therefore, the inventor discloses in this article by these means or other means (as by disappearance or the inhibition D factor or properdin) selectivity and has suppressed complement bypass, to suppress airway hyperreactivity and airway inflammation.The inventor shows that the B factor is to induce experimental asthma necessary.Importantly, the B factor is indispensable in the attack phase (or effector phase) of this model, and the monoclonal antibody of the selective binding B factor is sucked lung or has blocked the progress of the airway hyperreactivity relevant with allergic inflammatory diseases (AHR) and airway inflammation by whole body, as test as shown in the asthmatic model routine.In addition, the inventor finds that this inhibitory action obtains especially by suppressing complement bypass, and this is because other result shows in this model system, and C4 gene knockout (C4-/-) mice can suffer from AHR, and the B factor gene knocks out (fB-/-) mice and can not suffer from AHR.Therefore, the inventor find to suppress complement bypass (by any means) be suppress AHR and airway inflammation and treatment or prevention are relevant therewith thus disease and disease institute necessary and sufficient.In addition, the inventor shows that suppressing CCP is not that inhibition AHR or airway inflammation are necessary, therefore, as mentioned above, follows instruction of the present invention, can avoid the unwelcome result relevant with suppressing CCP.

The B factor antibody

Therefore, first embodiment of the present invention relates to antibody or its Fab that selectivity suppresses complement bypass, particularly B factor antibody.Equally, have mutually homospecific antigen-binding polypeptides and also be particularly preferred for the present invention.On the one hand, antibody be suppressed with the albumen of complement bypass or stop with another kind of protein bound mode (normal conditions be natural or physiological condition under these two kinds of albumen interact) albumen of this complement bypass of selective binding.On the other hand, antibody is suppressed with albumen or stops and activates another kind of proteic mode (generally both interact) this albumen of selective binding, though this albumen may to small part in conjunction with other albumen.The particularly preferred antibody and the Fab thereof that are used for selectivity inhibition complement bypass comprise B factor antibody, particularly this paper described herein mAb1379 antibody described in detail.

This paper describes in detail and for example understands the selective binding B factor and the antibody (and Fab) that suppresses complement bypass according to the present invention.In one embodiment, antibody or its Fab are combined in the conservative mating surface of (particularly in the mammalian species) in the animal species conservative this albumen (as the B factor) or epi-position (i.e. the albumen generation cross reaction of this antibody and two or more different mammalian species).Particularly, the present invention includes in conjunction with from least two kinds, the antibody of the B factor of preferred several different mammalian species, these mammals include but not limited to people, the primates except that the people, mice, rat, pig, horse and rabbit.Preferably, the present invention includes the antibody in conjunction with the B factor, this B factor is from people and another kind of at least animal species, and preferably another kind of at least mammalian species includes but not limited to primates, mice, rat, pig, horse and rabbit except that the people.In one embodiment, antibody or its Fab are in conjunction with the 3rd homology repetitive sequence (SCR) of the B factor.In one embodiment, antibody or its Fab are in conjunction with stoping the D factor to shear the zone of the B factor of the B factor.In one embodiment, antibody is monoclonal antibody.In one embodiment, antibody is 1379 alleged antibody of this paper (i.e. the antibody of being produced by the hybridoma cell line of identical numbering, ATCC preserving number PTA-6230) or its Fab.

Hybridoma 1379 described herein (or mAb1379) was preserved in American type culture collection (ATCC on the 21st in JIUYUE in 2004, be positioned at 10801University Blvd, Manassas, VA 20110-2209), this preservation center meets the clause of budapest treaty of the microbial preservation that is used for proprietary program of international recognition, and this hybridoma has obtained ATCC preserving number PTA-6230.

According to the present invention, its size of minimum dimension of albumen, a proteic part (as fragment, a part, domain etc.) or proteic zone or epi-position is enough to play the epi-position that forms as antibody or conservative mating surface or as the target material of analyzed in vitro.In one embodiment, albumen of the present invention be about at least 4,5,6,7 or 8 aminoacid (as in analysis, be suitable as antibody epitope or as detectable polypeptide) be about 25 aminoacid at least or be about 50 aminoacid at least be about 100 aminoacid at least or be about at least 150 aminoacid etc. any between 4 aminoacid to the total length that is up to albumen or its part or longer between whole integers length (as 8,9,10 ... 25,26......500,501......).

The well known coding people B factor and the gene of other complement protein and nucleotide sequence and these proteic aminoacid sequences of coding region.For example, the gene of the coding people B factor and other complement protein is numbered No.NG_000013 in ncbi database.The coded sequence of the B factor is numbered No.NM_001710 in ncbi database, the aminoacid sequence of B factor preproprotein is numbered No.NP_001701 or P00751 in ncbi database.The aminoacid sequence that ncbi database is numbered No.P00751 is people's preproprotein B factor sequence, is expressed as SEQ ID NO:1 in this article.The sequence from other animal species is also known in this area.By comparing, in mice B factor sequence, (for example number No.P04186 referring to ncbi database, be expressed as SEQ ID NO:6 in this article), Three S's CR domain is positioned at the preceding proteic 160-217 position of this 761 aminoacid, and sophisticated muroid B factor protein is crossed over the 23-761 position of SEQ ID NO:6.

Be expressed as albumen before the people B factor of SEQ ID NO:1 and be 764 amino acid whose albumen of signal peptide with leap amino acid/11-25.The 26-764 position of the corresponding SEQ ID of the ripe chain NO:1 of the B factor is expressed as SEQ ID NO:2 in this article.Three SCR zones of the people B factor are expressed as SEQ ID NO:3 (SCR1 in this article, also be Sushi 1, cross over SEQID NO:1 from about 35 to about 100, or cross over SEQ ID NO:2 from about 5 to about 75), SEQ ID NO:4 (SCR2, also be Sushi 2, cross over SEQ IDNO:1 from about 101 to about 160, or cross over SEQ ID NO:2 from about 76 to about 135) and SEQ ID NO:5 (SCR3, also be Sushi 3, cross over SEQID NO:1 from about 163 to about 220, or cross over SEQ ID NO:2 from about 138 to about 195).

Based on using the described fragment of Hourcade (referring to Hourcade, 1995, J.Biol.Chem.) epitope mapping that exemplary antibodies of the present invention is carried out (referring to embodiment), in a preferred embodiment, anti-B factor antibody preferred combination of the present invention epi-position or conservative mating surface in part Three S's CR domain or that contain part Three S's CR domain, more preferably in conjunction with comprising that at least a portion comprises the epi-position of the people B factor of the sequence of ripe B factor protein (SEQ ID NO:2) from about Try139 position to about Ser185 position, in conjunction with comprising that at least a portion comprises the epi-position of the people B factor of the sequence of ripe B factor protein (SEQ ID NO:2) from about Try139 position to about Ser141 position, in conjunction with comprising that at least a portion comprises the epi-position of the people B factor of the sequence of ripe B factor protein (SEQ ID NO:2) from about Glu182 position to about Ser185 position, in conjunction with comprising that at least a portion comprises the epi-position of the B factor of the people B factor (SEQ ID NO:2) of the equivalent position of any one or a plurality of down column position or non-human B factor sequence: Try139, Cys140, Ser141, Glu182, Gly184 or Ser185, or in conjunction with the epi-position of the B factor of the equivalent position that comprises the animal species of at least a portion except that the people.Those skilled in the art can be easily compares the sequence of the people B factor and the sequence from the B factor of other animal species, detects the specific part in the Three S's CR district of the position in SCR district and corresponding above-mentioned amino acid position.For example, BLAST2 sequence (the Tatusova and Madden that uses Tatusova and Madden to describe, (1999), " Blast 2 sequences-a newtool for comparing protein and nucleotide sequences ", FEMS Microbiollett.174:247-250 includes in herein by quoting in full) two particular sequences are compared each other.

Another epi-position model and collection of illustrative plates based on exemplary antibodies of the present invention, in another embodiment preferred, the epi-position of part Three S's CR domain in the part Three S's CR domain of the anti-B factor antibody preferred combination B factor of the present invention or that contain the B factor (conservative mating surface), this part comprises at least one or a plurality of following amino acid position or its equivalent position in non-human B factor sequence: the A137 of SEQ ID NO:2, Y139, S141, E182, S185, T189, E190 and S192.An aspect of of the present present invention, epi-position is arranged in the part Three S's CR domain of the B factor or contains the part Three S's CR domain of the B factor, this part comprises all or substantially all following amino acid position or its equivalent position in non-human B factor sequence: the Ala137 of (five, six or seven) SEQ IDNO:2 at least, Tyr139, Ser141, Glu182, Ser185, Thr189, Glu190 and Ser192.On the other hand, the epi-position of anti-B factor antibody identification of the present invention is arranged in the part Three S's CR domain of the B factor or contains the part Three S's CR domain of the B factor, this part is made up of following amino acid position or its equivalent position in non-human B factor sequence of SEQ IDNO:2: Ala137, Tyr139, Ser141, Glu182, Ser185, Thr189, Glu190 and Ser192.

In one embodiment, the epi-position of B factor antibody of the present invention identification also can more specifically be defined as the non-linear epi-position of three dimensional structure of the part of the Three S's CR domain that is arranged in the B factor.The part that comprises epi-position is the almost amino acid position Ala137-Ser192 of whole (as at least about 90%) of SEQ ID NO:2 or the determined B factor of the equivalent position three dimensional structure of non-human B factor sequence, and prerequisite is that this sequence is aligned to the conformation that natural total length B factor sequence occurs on conformation.The model of the three dimensional structure of the B factor has been illustrated the epi-position of mAb1379, for example can be referring to Figure 11 and 12.This paper employed proteic " three dimensional structure " or the arrangement of " tertiary structure " finger protein component in three-dimensional.This term is known by those skilled in the art.The representative in tangible medium of the three dimensional structure of term as used herein " model " finger protein, polypeptide or peptide.For example, model can be three dimensional structure in e-file, on the computer screen, the expression on the paper spare (promptly in two-dimensional medium) and/or be expressed as mallet figure.

According to the present invention, " epi-position " of given albumen or peptide or other molecule is generally defined as a macromolecular part relevant with antibody or the site on it, antibody or its Fab will be in conjunction with this part or sites, and can produce antibody at this part or site.The term epi-position can be exchanged with given albumen or antigenic term " antigenic determinant " " antibody combining site " or " conservative mating surface " and be used.More specifically, epi-position can be defined as participating in the amino acid residue of antibodies, also can be defined as three-dimensional conformation (as comformational epitope or conservative mating surface).Epi-position can be contained in little to the peptide of about 4-6 amino acid residue, maybe can be contained in the proteic big fragment, and at the three dimensional structure that refers to epi-position, when being meant the antibodies epi-position especially, epi-position need not to be made of the continuous amino acid residue.The antibodies epi-position is generally comformational epitope, rather than sequential epitope (being linear epitope), in other words, i.e. and the determined epi-position of amino acid residue of three-dimensional arrangement on bonded with it, albumen of antibody or the polypeptide surface.As mentioned above, comformational epitope be can't help the continuous sequence of amino acid residue and is constituted, but the substitute is, and this residue may extensively be separated in the one-level protein sequence, forms the 3-D natural conformation by protein folding, and this residue forms mating surface together.The epi-position that mAb1379 discerned is a comformational epitope, but not linear epitope.

Use known method, those skilled in the art can identify and/or assemble comformational epitope and/or sequential epitope, these methods comprise mutation analysis (as site directed mutagenesis), prevention Proteolytic enzyme edman degradation Edman (albumen footprinting method), use as synthetic peptide and pepscan, the mimic epitope analysis of BIACORE or ELISA, the antibody competition collection of illustrative plates, the combined peptide library screening, ground substance assistant laser parsing-ionization flight time (MALDI-TOF) mass spectrometry or threedimensional model are (as using any suitable software program, include but not limited to MOLSCRIPT 2.0 (Avatar Software AB, Heleneborgsgatan 21C, SE-11731 Stockholm, Sweden), graphic display program O (Jones et.al., Acta Crystallography, vol.A47, p.110,1991), graphic display program GRASP or graphic display program INSIGHT).For example, molecular replacement or other technology and the known three dimensional structure of associated protein three dimensional structure can be used, and comformational epitope can be inferred in conjunction with the antibody of this structure with the simulation B factor.In fact, can use the combination of a kind of or these technology in these technology, to determine the antibodies epi-position.Figure 11 and 12 illustrates the information that matched moulds is intended epitope analysis and mutation analysis of finishing, and uses threedimensional model to identify the epi-position of B factor antibody of the present invention.

Term used herein " selective binding " refers to that a kind of albumen combines with the specificity of another kind of albumen (as the binding partners of antibody, antibody fragment or conjugated antigen), wherein is significantly higher than the background contrast of analysis by what any standard method (as immunoassay) recorded statistically in conjunction with level.For example, when carrying out immunoassay, contrast generally comprises the reacting hole/pipe that only contains antibody or Fab (promptly not having antigen), and the amount of the wherein reaction of antibody or its Fab when no antigen (as non-specific bond in the hole) is considered to background.Can detect combination by the various standard methods of this area, these methods include but not limited to cell sorting (FACS) method and the flow cytometry of Western blotting, immunoblotting, Enzyme Linked Immunoadsorbent Assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surface plasma resonance technology, chemoluminescence method, fluorescence polarization method, phosphorimetry, immunohistochemical analysis, ground substance assistant laser parsing-ionization flight time (MALDI-TOF) mass spectrometry, microscopic cells art, microarray, microscopy, fluorescence-activation.

One embodiment of the invention comprise antibody or its Fab, and it is the competitive inhibitor of the B factor in conjunction with anti-B factor antibody (as monoclonal antibody 1379).According to the present invention, the B factor in conjunction with the competitive inhibitor of anti-B factor antibody of the present invention be with the identical epi-position of known anti-B factor antibody of the present invention (as mAb1379) or similar epi-position inhibitor (as another kind of antibody or Fab or polypeptide) in conjunction with the B factor, therefore known anti-B factor antibody is suppressed with combining of the B factor.Competitive inhibitor can be compared bigger affinity in conjunction with target material (as the B factor) with anti-B factor antibody.Can described herein and anti-B factor antibody 1379 similar modes (as suppressing complement bypass, suppressing ischemic injuries of perfusion again of the airway hyperreactivity of animal, the airway inflammation that suppresses animal, inhibition animal or the like) use competitive inhibitor.For example, one embodiment of the invention relate to separation antibody or its Fab of specificity in conjunction with the B factor, wherein this antibody or its fragment competitive inhibition mAb1379 specificity are in conjunction with the B factor, and wherein when this antibody or its fragment during in conjunction with the B factor, complement bypass is suppressed, perhaps, the ability of mAb1379 inhibition complement bypass is suppressed.Another embodiment relates to separation antibody or its fragment of specificity in conjunction with the B factor, and wherein anti-or its fragments specific of this separation antibody or its fragment competitive inhibition two is in conjunction with the B factor, and wherein this two anti-or its fragment in conjunction with the Three S's CR domain of the B factor.

Can use standard method (as competitive ELISA or other binding analysis) the being at war with property analysis of this area.For example, the bonded ability of the anti-B factor antibody (as mAb1379) by suppressing the B factor and known labelling can detect competitive inhibitor and it is carried out quantitatively.Antibody when the people B factor exists-antibody competition analysis is for example being described among the embodiment 3.The competitive inhibitor of the B factor in conjunction with the anti-B factor 1379 described in embodiment 3 and the table 4.

According to the present invention, antibody is characterised in that they comprise immunoglobulin domains, so they are members of proteic immune globulin albumin extended familys.Generally speaking, antibody molecule comprises two types chain.Wherein a kind of chain refers to heavy chain or H chain, and another kind of chain refers to light chain or L chain.Two kinds of chains with etc. mol ratio exist, and each antibody molecule generally has two H chains and two L chains.Article two, the H chain links together by disulfide bond, and every H chain and every L chain link together by disulfide bond.Have only two types L chain, be lambda (λ) chain and kappa (κ) chain.On the contrary, five kinds of main H chains that are called as isotype are arranged.This five class comprises IgM (IgM or μ), immunoglobulin D (IgD or δ), immunoglobulin G (IgG or λ), immunoglobulin A (IgA or α) and IgE (IgE or ε).Distinctive feature between these isotypes is determined by the constant region of immunoglobulin and is gone through hereinafter.Human normal immunoglobulin's molecule comprises four kinds of subclass (comprising IgG1 (γ 1), IgG2 (γ 2), IgG3 (γ 3) and IgG4 (γ 4)) of nine kinds of isotype: IgM, IgD, IgE, IgG and two kinds of subclass (comprising IgA1 (α 1) and IgA2 (α 2)) of IgA.People's IgG subclass 3 and IgM are the most effective complement activation agent (classical complement system), and IgG subclass 1 and subclass 2 (degree is lower) are the activator of the middle minuent of classical complement system.The IgG4 subclass is activating complement system (classical pathway or alternative pathway) not.Unique human normal immunoglobulin's isotype of known activating complement bypath system is IgA.In mice, the IgG subclass is IgG1, IgG2a, IgG2b and IgG3.Muroid IgG1 is activating complement not, and IgG2a, IgG2b and IgG3 are the complement activation agent.

Every the H chain or the L chain of immunoglobulin molecules comprise two zones, are called L chain variable region (V _LDistinguish) and L chain constant region (C _LDistinguish), and H chain variable region (V _HDistinguish) and H chain constant region (C _HThe district).Complete C _HThe district comprises three subprovinces (CH1, CH2, CH3) and a hinge region.Generally speaking, H chain and L chain can form the arm of the immunoglobulin molecules with immune globulin variable region.Complete immunoglobulin molecules comprises two continuous (connecting as disulfide bond) arms.Therefore, each arm of whole immunoglobulin comprises V _H+LDistrict and C _H+LThe district.Term as used herein " variable region " or " V district " refer to V _H+LThe district (is also referred to as F _vFragment), V _LDistrict or V _HThe district.Term " constant region " or " the C district " that uses in this article refers to C in addition _H+LDistrict, C _LDistrict or C _HThe district.

With protease immunoglobulin is carried out restrictive diges-tion and can produce two kinds of fragments.Fab refers to Fab, Fab ' or F (ab ') ₂Fragment.The fragment that lacks the ability of conjugated antigen refers to F _cFragment.The Fab fragment comprises and containing and V _HDistrict and portion C _HL chain (the V that district (CH1 district) is complementary _L+ C _LAn arm of the immunoglobulin district).Fab ' fragment correspondence has the Fab fragment of the part hinge region that adheres to the CH1 district.F (ab ') ₂Fragment is corresponding passes through disulfide bond two covalently bound each other Fab ' fragments usually at hinge region usually.

C _HThe district has determined the isotype of immunoglobulin, and gives different functional characters according to different isotypes.For example, the μ constant region can form the pentamer of IgM molecule, and the α constant region can form dimer.

The antigenic specificity of immunoglobulin molecules is given by the aminoacid sequence in variable region or V district.Therefore, the V district of different immunoglobulin molecules can be significantly different according to its antigenic specificity.Some part in V district is more conservative than other parts, is called as skeleton district (FW district).On the contrary, some part alterable height in V district is called as the hypervariable region.V _LAnd V _HWhen matched in immunoglobulin molecules in the district, each regional hypervariable region constituted jointly the hypermutation ring (hypervariable loop) that forms antigen binding site.Therefore, the hypermutation ring has determined the specificity of immunoglobulin, is called as complementary determining region (CDR), and this is because its surface is complementary with antigen.

Other transmutability in V district is given by the combination transmutability of the constant gene segment C in coding immunoglobulin V district.It is constant gene segment C that immunoglobulin gene comprises multiple, and this section is reset the rearrangement immunoglobulin gene that forms the coding immunoglobulin molecules in vivo.L chain V constant gene segment C and J constant gene segment C (jointing) coding V _LThe district.H chain V constant gene segment C, D constant gene segment C (changeable section) and J constant gene segment C (jointing) coding V _HThe district.

L chain and H chain V constant gene segment C all contain and have variable three zones of primary amino acid sequence.This zone refers to L chain CDR1, CDR2 and CDR3 and H chain CDR1, CDR2 and CDR3 respectively.The length of L chain CDR1 can be at different V _LDistrict's significant change.For example, the length of CDR1 can change between about 17 aminoacid at about 7 aminoacid.On the contrary, the length of L chain CDR2 and CDR3 is generally at different V _LThe district does not change.The length of H chain CDR3 can be at different V _HDistrict's significant change.For example, the length of CDR3 can change between about 20 aminoacid at about 1 aminoacid.The flanking region in each H chain and L chain CDR district is the FW district.

Other function aspects of immunoglobulin molecules comprises the coordination valence of immunoglobulin molecules, the affinity of immunoglobulin molecules and the affinity of immunoglobulin molecules.The employed affinity of this paper refers to the intensity (as unit price Fab fragment in conjunction with monovalent antigen) of the immunoglobulin molecules conjugated antigen on the single site of immunoglobulin molecules.Affinity is different from affinity, and the latter refers to the summation of the intensity of immunoglobulin conjugated antigen.The measurement of immunoglobulin binding affinity can be by using the standard technique of this area, as competitive combination technology, equilibrium dialysis or BIAcore method.The employed coordination valence of this paper refers to the different antigen binding site numbers (being antigen binding site number on the antibody molecule of each Fab) of each immunoglobulin molecules.For example, the unit price immunoglobulin molecules simultaneously can only be in conjunction with an antigen, and the bivalence immunoglobulin molecules can be simultaneously in conjunction with two or more antigens etc.The proteic unit price and the bivalent antibody of selective binding complement bypass all are contained in herein.

In one embodiment, antibody is bi-specific antibody or multi-specificity antibody.As bivalence (or multivalence) antibodies antigen, bispecific (or polyspecific) antibody capable is in conjunction with two (or a plurality of) antigens, but in this case, antigen is different antigen (being that antibody shows bispecific or more specificity).For example, the proteic antibody in the selective binding complement bypass of the present invention (anti-as described in this article B factor antibody) can be built into bi-specific antibody, and wherein second antigen-binding specificity is at required target material.Therefore, a kind of bi-specific antibody that the present invention comprised comprises the antibody with following composition: (a) albumen in the conjugated complement bypass (as the B factor) first (as first antigen-binding portion thereof); (b) in conjunction with the second portion of the cell surface molecule of cellular expression.In this embodiment, second portion can be in conjunction with any cell surface molecule.A kind of preferred cell surface molecule is receptor or part, so the specific cell or tissue type of this antibody target and/or give the intravital specific site of animal of antibody.In one embodiment, second antigen-binding specificity is at complement receptors.A kind of particularly preferred complement receptors includes but not limited to II type complement receptors (CR2).Therefore selective binding CR2 also can be used for the antibody of this embodiment of the present invention is for example describing in the U.S. Patent No. 6,820,011.

In one embodiment, antibody of the present invention comprises humanized antibody.Humanized antibody is a kind of like this molecule, and its antigen binding site is from the immunoglobulin of the species except that the people, and the part in the remaining immunoglobulin source of this molecule derives from the human normal immunoglobulin.Antigen binding site can comprise the complete variable region that is fused to human constant region or only comprise grafting complementary determining region (CDR) in the suitable human skeleton district in the variable region.Humanized antibody can be produced (vide infra) by for example analog antibody variable region and by using gene engineering (as the CDR grafting) to produce antibody.Found description, for example referring to Morrison et al. (1984) Proc.Natl.Acad.Sci.USA 81:6851-55 to the technology of multiple production humanized antibody; Whittle et al. (1 987) Prot.Eng.1:499-505; Co et al. (1990) J.Immunool.148:1149-1154; Co et al. (1992) Proc.Natl.Acad.Sci.USA 88:2869-2873; Carter et al. (1992) Proc.Natl.Acad.Sci.89:4285-4289; Routledge et al. (1991) Eur.J.Immunol.21:2717-2725 and PCT patent publication WO 91/09967, WO91/09968 and WO 92/113831.

Separation antibody of the present invention can comprise the serum that contains this antibody or antibody purified in various degree.Whole antibody of the present invention can be monoclonal antibody or polyclonal antibody.Perhaps, the present invention can use following antibody: whole function equivalents of antibody, as one or more antibody structures territory by the Fab of truncate or disappearance (as Fv, Fab, Fab ' or F (ab) ₂Fragment) and genetically engineered antibody or its Fab, comprise single-chain antibody, humanized antibody (as mentioned above), in conjunction with more than the antibody (as bi-specific antibody) of an epi-position or can be in conjunction with the antigenic antibody of one or more differences (as bi-specific antibody or multi-specificity antibody).

Genetically engineered antibody of the present invention comprises the antibody of producing by the standard recombinant dna technology, and these technology relate to the controlling and expressing of DNA of encoding antibody variable region and/or constant region.Concrete example comprises the chimeric antibody (V of antibody _HDistrict and/or V _LDistrict and remaining antibody moiety are from separate sources) and CDR grafted antibody (and Fab), wherein at least one CDR sequence and randomly at least one variable region skeleton aminoacid come from a kind of source and remaining variable region and constant region partly when suitable () come from separate sources.For example being structured in of chimeric antibody and CDR grafted antibody described among European patent application EP-A0194276, EP-A0239400, EP-A0451216 and the EP-A0460617.

In one embodiment, chimeric antibody produced according to the invention, this antibody comprise the antibody variable region of the albumen (as the B factor) of conjugated complement bypass, and merge with this district, as the albumen of second targeting moiety.For example, it is relevant with the cell or tissue of targeting that targeting moiety can comprise, or with the relevant albumen of particular system in the animal.For example, targeting moiety can be a part of complement receptors.A kind of preferred complement receptors that is used for this respect of the present invention comprises II type complement receptors (CR2).In fusion rotein or chimeric protein, use CR2 and part thereof (as sending delivery system) in U.S. Patent No. 6,820, describe in detail in 011.

Generally speaking, in the production of antibody, antigen (can produce required antibody at this antigen) is exposed to suitable laboratory animal, such as but not limited to rabbit, sheep, hamster, Cavia porcellus, mice, rat or chicken.Generally speaking, by being injected to animal, the antigen of effective dose comes immune animal.Antigenic effective dose refers to that induced animal produces the needed amount of antibody.Then, the immune system of animal can react in predetermined a period of time.Immunologic process can be carried out repeatedly up to finding that immune system produces at antigenic antibody.For obtaining specificity, collect the serum (with regard to chicken, antibody can be collected) of the animal that contains required antibody from egg at antigenic polyclonal antibody.This serum can be used as reagent and uses.Polyclonal antibody can be by for example handling purification from serum (or egg) with ammonium sulfate.

Monoclonal antibody can be produced according to the method (Nature 256:495-497,1975) of Kohler and Milstein.For example, collect bone-marrow-derived lymphocyte, merge with the myeloma cell then from the spleen (or any suitable tissue) of immune animal, with obtain can be in proper culture medium the hybridoma group of continuous growth.The hybridoma of producing required antibody can screen by the required antigenic ability of antibodies that detects hybridoma production.

The preferable methods of producing antibody of the present invention comprises that (a) gives the albumen of animal effective dose or peptide (as the B factor protein or contain the peptide of its domain) producing antibody, and (b) reclaims antibody.In another approach, antibody of the present invention passes through recombinant production.For example, in case obtain to express the cell line of antibody of the present invention,, can comprise the sequence of the CDR that encodes from wherein cloning the variable region gene of cDNA and the required antibody of energy identification code so as hybridoma.Therefore, antibody of the present invention or Fab can obtain by one or more reproducible expression vectors of preparation and with its conversion/transfection proper host cell, and in this host cell, produce antibody, wherein said expression vector contains the DNA sequence of the variable region of encoding antibody heavy chain or light chain at least, and randomly contains other DNA sequence of the remaining part of required heavy chain of coding and/or light chain.Suitable expressive host comprises antibacterial (as coli strain), fungus (yeast particularly, member as pichia (Pichia), Saccharomyces (Saccharomyces) or Kluyveromyces (Kluyveromyces)) and mammal cell line, as nonproductive type myeloma cell line, as mice NSO system or Chinese hamster ovary celI.For obtaining effectively to transcribe and translate, the DNA sequence in each carrier should comprise suitable regulating and controlling sequence, particularly effectively is connected to promoter and targeting sequencing on the variable region sequences.Produce the concrete grammar of antibody generally is familiar with and conventional the use by this approach.For example, the base molecule biological method is described (Molecular Cloning, Cold Spring Harbor Laboratory, NewYork, 1989) by people such as Maniatis; Can be according to people's such as Sanger description (PNAS 74, 5463, (1977)) and Amersham International plc order-checking handbook carry out dna sequencing, and can be according to people's such as Kramer method (Nucl.Acids Res. 12, 9441, (1984)) and the handbook of AnglianBiotechnology Ltd. can carry out site directed mutagenesis.In addition, there is the publication that much comprises patent specification to describe in detail to be fit to the technology for preparing antibody by the suitable cell of controlling DNA, construction of expression vector and conversion, for example as Mountain A and Adair, JR is at " biotechnology and genetic engineering summary " (Biotechnology and GeneticEngineering Reviews) (ed.Tombs, M P 10, Chapter 1,1992, Intercept, Andover, UK) and sum up in the european patent application mentioned in front like that.

Other for example adopts that the method or the lymphocyte antibody method among the selected US5627052 of display technique of bacteriophage (referring to for example US5969108, US5565332, US5871907, US5858657) also can be used for producing antibody of the present invention and/or antigen fragment, and this is very apparent for the technical staff.

Therefore, another aspect of the present invention relates generally to be used for compositions and the method that selectivity suppresses the complement bypass of animal, this animal suffers from complement bypass and activates disease or the disease that works or this disease risk (progress that helps disease or disease as the activation of complement bypass of suffering from is arranged, increased the weight of at least one symptom of this disease or disease, perhaps caused disease or disease).This method comprises uses novel B factor antibody of the present invention (above describing in detail).This disease or disease include but not limited to the disease relevant with airway hyperreactivity (comprising the relevant airway hyperreactivity of inflammation), ischemia reperfusion injury and fetal loss.Compositions and the preparation that detailed description is contained this antibody and Fab thereof and simulate the specific antigen-binding polypeptides of B factor antibody described herein, and the discussion that gives method and dosage below.

The method of prevention or inhibition airway hyperreactivity and airway inflammation

Complement bypass based on the inventor is to suppress airway hyperreactivity and necessary and sufficient this discovery of airway inflammation, and another embodiment of the present invention relates to and suppresses to suffer from inflammation relevant airway hyperreactivity or airway inflammation or the airway hyperreactivity of the animal that suffers from this disease risk and/or the method for airway inflammation are arranged.This method comprises that selectivity suppresses the step of suffering from airway hyperreactivity or the complement bypass of the animal that suffers from this disease risk being arranged, described airway hyperreactivity comprise inflammation be correlated with airway hyperreactivity (appearance that is airway hyperreactivity is because inflammation or inflammatory process, perhaps with air flue in inflammation occur simultaneously or occur prior to inflammation).

Discussion hereinafter has been described in detail treatment or the prevention airway hyperreactivity of animal and/or airway inflammation or relevant therewith disease or disease aspect.But, it should be understood that in any embodiment that may be used on invention described herein to inhibitor, route of administration, dosage, treatment indicator, to the generality discussion of description of preparation or the like (promptly at other disease or disease beyond those diseases relevant or the disease) with airway hyperreactivity and airway inflammation.For example, hereinafter many summaries aspect of described invention may be used on the specific inhibition of complement bypass, to treat other disease, as ischemia reperfusion injury.

According to the present invention, the complement bypass that suppresses animal refers to suppress at least a proteic expression and/or the biological activity as the part of complement bypass.This albumen includes but not limited to the B factor, the D factor or properdin." selectivity " suppresses complement bypass and means that method of the present invention preferentially or uniquely suppresses complement bypass, but do not suppress or significantly do not suppress at least other approach (comprising CCP or lectin pathway) of complement activation.For example, novel B factor antibody of the present invention and Fab thereof are the example that selectivity suppresses the reagent of complement bypass.This definition is applied in other method described herein, and wherein complement bypass is suppressed by selectivity.

The inhibition of complement bypass of the present invention can be finished by the protein expression (transcribe or translate) or the biological activity that directly influence complement bypass, or by direct influence the albumen of protein binding complement bypass or influence the ability that albumen otherwise helps to activate the complement in the alternative pathway finish.More specifically, in one embodiment, the transcribing or proteic translation of proteic expression finger protein.Therefore, method of the present invention can suppress proteic in this proteic animal of natural expression and transcribe and/or translate (as by giving reagent that Profilin expresses and animal being carried out genetic modification to reduce protein expression).In another embodiment, be suppressed at any measurable (detectable) that be defined as this pathway activities herein of complement bypass reduces (promptly reduce, reduce, suppress), for example passes through protein expression and/or bioactive measurable reduction of complement bypass.

According to the present invention, " airway hyperreactivity " or " AHR " refers to the unusual of air flue, and it makes air flue be highly susceptible to narrowing down and/or excessively narrowing down to the stimulation that can cause flow limitation.AHR can be the functional change of respiratory system, and this is because inflammation in the air flue (being the relevant AHR of inflammation) or because airway remodeling (as passing through collagen deposition).Flow limitation is meant irreversible or reversible the narrowing down of air flue.Flow limitation or airway hyperreactivity can be caused by following phenomenon: the interior and wellability disease on every side of unusual and air flue of the change of the destruction of collagen deposition, bronchospasm, airway smooth muscle hypertrophy, airway smooth muscle contraction, mucous secretion, cell deposition, epithelium, the change of epithelium penetrance, smooth muscle function or sensitivity, pulmonary parenchyma.Although AHR is the symptom different with inflammation, still have in these inducements many relevant with inflammation (be that AHR is particular disorder as described above or symptom, it can (but not always) with air flue early stage inflammation or concurrent inflammation relevant).By being exposed to exciting agent or stimulus object (be also referred to as AHR in this article and excite stimulus object), AHR can excite in the patient with disease relevant with above-mentioned inducement.This stimulus object includes but not limited to anaphylactogen, acetonyl choline, histamine, leukotriene, saline, ventilation enhancing, motion, sulfur dioxide, adenosine, propranolol (propranolol), cold air, antigen, Kallidin I, acetylcholine, prostaglandin, ozone, environmental air pollution thing and composition thereof.The present invention relates to and the relevant airway hyperreactivity of any breathing disease with inflammation, the particularly airway hyperreactivity of allergen-induced.

Airway hyperreactivity is relevant with the inflammation of allergic inflammation and/or virus induction usually.The airway hyperreactivity relevant with allergic inflammation can appear to be suffered from the disease that includes but not limited to chronic obstructive airway disease or has among the patient who suffers from this disease risk.This disease includes but not limited to: asthma, chronic obstructive disease of lung, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis, the eosinophilic granulocyte pneumonia, emphysema, bronchitis, allergic bronchitis, bronchiectasis, cystic fibrosis, tuberculosis, hypersensitivity pneumonitis, occupational asthma, sarcoidosis, RADS, interstitial lung disease, hypereosinophilic syndrome, rhinitis, sinusitis, exercise-induced asthma, asthma and parasitic disease of lung are brought out in pollution.The airway hyperreactivity relevant with the inflammation of virus induction appears at by viral infection or has in the patient of infected risk, and these viruses include but not limited to respiratory syncytial virus (RSV), parainfluenza virus (PIV), rhinovirus (RV) and adenovirus.Use other disease of method of the present invention and reagent treatment or disease to comprise to relate to the inflammation that disease (as the general autoimmune disease) causes and/or any pulmonary disease or the pulmonary complication of airway hyperreactivity.For example, use the present invention can treat the pulmonary complication in the systemic lupus erythematosus.

The feature of inflammation generally is the release of inflammatory mediator (as cytokine or chemotactic factor), and this inflammatory mediator will participate in the cell of inflammation and raise to tissue.Airway inflammation is for appearing at the inflammation in the animal air flue (lung tissue, breathing cell and tissue).Disease relevant with allergic inflammation or disease are a kind of like this disease or disease, promptly in animal, bring out at a para-immunity of sensitizer (as anaphylactogen) and reply the release that (as Th2 type immunne response) can cause inflammatory mediator, inflammatory mediator is raised the cell that participates in inflammation, its existence can cause histologic lesion, and causes death sometimes.As mentioned above, AHR common relevant with airway inflammation (occur simultaneously with airway inflammation, perhaps may cause) by airway inflammation.It should be noted: the symptom of AHR or disease can not rely on inflammatory symptom treatment sometimes, vice versa (may be influential as treatment to inflammation to AHR, also may not have-they are diseases independently).

AHR can pass through stress test (stress test) and detect, and this test comprises the respiratory system function that the detection animal is replied exciting agent (being stimulus object).AHR can the relative baseline of respiratory function the form of change curve chart that the dosage of exciting agent is done measure (describing among the method for this detection and the employed mammal model embodiment hereinafter).Respiratory function (and biological characteristic of AHR) can detect by following manner, for example spirometry, plethysmography, peak flow, symptom score, sign (being breathing rate), roar toot, use, cough and the blood gas of motion tolerance, rescue medicament (being bronchodilator).The change that people's spirometry is measured respiratory function with exciting agent such as acetonyl choline or histamine.Deeply breathe by asking for help and with gas maximum duration, maximum, force, be blown in the quantifier of measuring air-flow and volume people's vital capacity is measured the most apace.The volume of air of breathing out in known first second is forced expiratory volume (FEV ₁), the cumulative volume of breath is forced vital capacity (FVC).Can obtain people's normal expected FEV ₁And FVC, and it is carried out standardization according to body weight, height, sex and race.The FEV of diseased individuals not ₁With FVC be specific people the normal expected value at least about 80%, and FEV ₁The ratio of/FVC is at least about 80%.This numerical value is measured in (quiescent condition of promptly representing the patient) back (promptly representing the lung resistance state that the patient is higher) before sucking exciting agent respectively.The position of resulting curve shows the sensitivity of air flue to exciting agent.

The dosage of exciting agent or the increase of concentration to influence of Pulmonary Function by measuring the animal attacked with exciting agent at first second forced expiratory volume (FEV ₁) and FEV ₁Than forced vital capacity (FEV ₁/ FVC ratio) detects.Cause people's FEV ₁The dosage or the concentration (PC of the exciting agent (being acetonyl choline or histamine) of decline 20% ₂₀FEV ₁) be the index of the degree of AHR.Use the known method of those skilled in the art can detect FEV ₁With the FVC value.

Can detect lung functions by measurement transpulmonary pressure (pressure differential between airway open and the body plethysmograph) and measure airway resistance (R _L), Cdgn dyanamic compliance (C _L) and high response.Volume is that the pressure that is calculated in the body plethysmograph changes, and air-flow is the digital difference of volume signals.Use method well-known to those skilled in the art (as by using the recursive least square scheme of moving equilibrium) to obtain resistance (R _L) and compliance (C _L).It should be noted and measure airway resistance (R in the mammal (as mice) except that the people _L) value can be used for diagnosing airflow obstruction (airflow obstruction), this is with the FEV that measures the people ₁And/or FEV ₁/ FVC ratio is similar.

Can use multiple exciting agent to measure the AHR value.Suitable exciting agent comprises directly and the indirect stimulation thing, and is generally the exciting agent that excites AHR in the body.The employed phrase of this paper " exciting agent " can exchange with phrase " AHR excites stimulus object " and use.Preferred exciting agent or stimulus object comprise for example anaphylactogen, acetonyl choline, histamine, organic stimulus object, irritative gas and chemical drugs, leukotriene, saline, ventilation enhancing, motion, sulfur dioxide, adenosine, propranolol, cold air, antigen, Kallidin I, acetylcholine, prostaglandin, ozone, environmental air pollution thing and composition thereof.Preferably, the tentative of AHR induced, acetonyl choline (Mch) is as exciting agent.The preferred concentration that is used for the Mch of concentration-response curve arrives between about 100 milligrams every milliliter (mg/ml) between about 0.001.The more preferably concentration that is used for the Mch of concentration-response curve arrives between about 50mg/ml between about 0.01.The more preferably concentration that is used for the Mch of concentration-response curve arrives between about 25mg/ml between about 0.02.When Mch was used as exciting agent, the degree of AHR was defined as causing the FEV of animal ₁What descend 20% needed Mch excites concentration (PC _{20 acetonyl choline}FEV ₁).For example, in the people, and use the also standard method in field, a normal person's PC _{20 acetonyl choline}FEV ₁Generally greater than 8mg/ml Mch.Therefore, in the people, AHR is defined as PC _{20 acetonyl choline}FEV ₁Less than 8mg/ml Mch.

According to the present invention, respiratory function can be by various static test assessments, and this test comprises the respiratory system function of detection animal when not having exciting agent.The example of static test comprises for example use, blood gas and the cough of spirometry, plethysmography, peak flow, symptom score, sign (being breathing rate), wheezing, motion tolerance, rescue medicament (being bronchodilator).Can in static test, assess lung functions by measuring following index, the electrical conductivity (SGL) of total lung capacity (TLC), TGV (TgV), pulmonary function residual volume (FRC), residual volume (RV) and lung volume for example, diffusion capacity for carbon monoxide of lung (DLCO), arterial blood gas comprise the pH, the P that are used for gas exchange _O2And P _CO2FEV ₁And FEV ₁/ FVC all can be used for detecting flow limitation.If in the people, use spirometry, individual's FEV ₁Can with FEV ₁Predicted value compare.FEV ₁Predicted value can obtain by age, sex, body weight, height and the ethnic standard normogram based on animal.Intact animal's FEV ₁Be generally the expectation FEV of animal ₁At least about 80%.Flow limitation causes FEV ₁Or FVC is less than 80% of predicted value.The another kind of method of flow limitation that detects is with FEV ₁Ratio (FEV with FVC ₁/ FVC) be the basis.The individuality of the disease that do not take a disease is defined as its FEV ₁The ratio of/FVC is at least about 80%.Airflow obstruction causes FEV ₁The ratio of/FVC drops to less than 80% of predicted value.Therefore, the animal with flow limitation is defined as its FEV ₁/ FVC is less than about 80%.

The employed reduction airway hyperreactivity of this paper refers to that any measurable reduction in the airway hyperreactivity and/or airway hyperreactivity appear at occurrence rate among the patient or any reduction of frequency.The reduction of AHR can be measured by using any above-mentioned technology or any other suitable method known in the art.Preferably, airway hyperreactivity or possible airway hyperreactivity are lowered, and the best is lowered to animal and no longer stands by airway hyperreactivity and cause or associated uncomfortable and/or changing function.The prevention airway hyperreactivity refers to can be prevented or end inducing of airway hyperreactivity before obviously detecting or measuring in the patient at the biological property of airway hyperreactivity as herein described.In case the biological property of one or more airway hyperreactivities can obviously be detected or measure, the airway hyperreactivity of Acute onset is considered to occur.

In one embodiment, method of the present invention has reduced the reactivity of the acetonyl choline of animal.Preferably, method of the present invention causes the PC of animal _{20 acetonyl choline}FEV ₁Value improves, therefore the PC that (this moment, animal excited with first concentration of acetonyl choline) obtained before using method of the present invention _{20 acetonyl choline}FEV ₁Value and the PC that (this moment, animal excited with first concentration that is twice in the acetonyl choline) obtained after using method of the present invention _{20 acetonyl choline}FEV ₁Be worth identical.Preferably, method of the present invention causes the PC of animal _{20 acetonyl choline}FEV ₁Value improves, therefore, and the PC that (this moment, animal excited to the acetonyl choline of about 8mg/ml with about 0.01mg/ml) obtained before using method of the present invention _{20 acetonyl choline}FEV ₁Value and the PC that (this moment, animal excited to the acetonyl choline of about 16mg/ml with about 0.02mg/ml) obtained after using method of the present invention _{20 acetonyl choline}FEV ₁Be worth identical.

In another embodiment, method of the present invention is with the FEV of animal ₁Improve animal and estimated FEV ₁At least about 5%, more preferably improved about 6% to about 100%, more preferably improved about 7% to about 100%, more preferably improved about 8% to about 100%.In another embodiment, method of the present invention is with the FEV of animal ₁Improved at least about 5%, preferably at least about 10%, more preferably at least about 25%, more preferably at least about 50%, more preferably at least about 75%.

In another embodiment, method of the present invention causes the PC of animal _{20 acetonyl choline}FEV ₁PC with respect to the intact animal _{20 acetonyl choline}FEV ₁Improved about double strength.The intact animal refers to knownly do not suffer from unusual AHR or to its insensitive animal.Patient or tested animal refer to doubtfully suffer from unusual AHR or to its responsive animal.

Therefore, the animal that suffers from airway hyperreactivity for example can be by using the animal that the above-mentioned wherein a kind of method that is used to measure airway hyperreactivity is measured or detect for its airway hyperreactivity, wherein as mentioned above, airway hyperreactivity generally excites stimulus object to be induced by being exposed to AHR.Equally, the animal that suffers from the airway hyperreactivity of allergen-induced for example can be by using the animal that the above-mentioned wherein a kind of method that is used to measure airway hyperreactivity is measured or detect for its airway hyperreactivity, wherein, airway hyperreactivity can be by being exposed to allergen-induced.In order to excite stimulus object such as allergen-induced by AHR, airway hyperreactivity can directly or indirectly cause (as being caused by the latter, being the latter's symptom, index, perhaps the incident that occurs simultaneously with the latter) significantly by being exposed to stimulus object.The symptom of AHR or biological property include but not limited to altered respiratory function (referring to above describing in detail)) index, breathing rate change, roar toot, the motion tolerance, cough and the altered blood gas that have reduced.The detection of any one or a plurality of these symptoms or measurement are the indexs of acute AHR outbreak.

With regard to anaphylactogen, airway hyperreactivity, directly or indirectly initiation obvious by the anaphylactogen of previous sensitization animal.One or many was exposed to anaphylactogen before sensitization referred to anaphylactogen, occurred the immunne response at this anaphylactogen thus.Individually do not occur when being exposed to anaphylactogen for the first time new relevant with anaphylaxis the replying (as histamine release, rhinitis, edema, vasodilation, bronchus compression (bronchial constriction) or airway hyperreactivity, airway inflammation), but in a single day produced cellullar immunologic response and humoral immunoresponse(HI) at anaphylactogen, individual just to this anaphylactogen " sensitization ".Then, when the sensitization individuality is exposed to identical anaphylactogen (attacking as anaphylactogen) once more, anaphylaxis will appear.In case it is individual to the anaphylactogen sensitization, the anaphylaxis meeting is exposed to anaphylactogen at every turn and becomes serious along with ensuing, not only produced allergic symptom because expose again at every turn, also further improved produce at anaphylactogen antibody level and at the level of the t cell response of anaphylactogen.

Generally speaking, with at the inflammation of relevant its feature of disease of the anaphylaxis of antigen (being anaphylactogen) at least partially in lung tissue.These diseases or disease are described hereinbefore.Effect of the present invention it should be noted: although may relate to the inhibition of irritated inflammation, but the present embodiment of the present invention at the treatment of AHR, does not therefore need to cause that the associated conditions of AHR such as irritated inflammation or inducement can significantly reduce or " healing " especially.Method of the present invention even after the inflammatory reaction of the pulmonary of animal is established fully, can reduce AHR completely effectively.There is the animal that suffers from the airway hyperreactivity risk to excite stimulus object or have and be exposed to this stimulus object risk, but do not show the animal of the feature of measuring or detecting or the symptom (those symptoms of in preamble, having described) of airway hyperreactivity as yet for being exposed to the AHR that is enough to cause AHR.The animal that the airway hyperreactivity risk of suffering from allergen-induced arranged by the anaphylactogen sensitization and be exposed to the amount (being the initiation amount or the challenging dose of anaphylactogen) of the anaphylactogen that is enough to cause AHR or the risk that is exposed to is wherein arranged, but does not show the feature that detects or measure of airway hyperreactivity or the animal of symptom for earlier as yet.There is the animal that suffers from the airway hyperreactivity risk also to comprise to be considered to easily suffer from this disease or disease or to its responsive animal.

Method of the present invention also can suppress or reduce the airway inflammation of animal.Inflammation, particularly acidophilia's inflammation are the signs that comprises the various respiratory disease of asthma.By using Several Parameters can assess airway inflammation, this Several Parameters includes but not limited to the accumulation (as eosinophilic granulocyte, macrophage, neutrophil cell, lymphocyte) of inflammatory cell in the lung, the variation that level changes and/or the interior mucus of lung generates of the various kinds of cell factor (as IL-4, IL-5, IL-10, IL-12, IL-13 and IFN-γ) in the bronchoalveolar lavage fluid (BALF).The measuring method of multiple these parameters has been shown among the embodiment.

Reagent of the present invention and preparation can give any animal patient, preferred administration of human.According to the present invention, can be used for of reagent or preparation, suppressed AHR, airway inflammation or the treatment disease relevant with these diseases.The patient candidate that is fit to of method of the present invention includes but not limited to suffer from this disease or disease or the patient who suffers from this disease risk (as being easy to suffer from this disease) is arranged.Although effect of the present invention may relate to the remarkable treatment benefit to the patient, but as mentioned above, the present invention relates generally to the treatment of AHR and/or airway inflammation, therefore do not need to cause the disease or the inducement of AHR or airway inflammation, or the disease relevant with these diseases is reduced significantly or " healing ".This notion also is widely used in other disease or disease that complement bypass works.

Therefore, the treatment benefit is not necessarily cured specified disease or disease (comprising any disease described herein or disease), generally includes following result most but preferably include: alleviate disease or disease, eliminate a disease or secondary disease or disease and/or prevent disease or disease that disease, symptom, prevention or alleviation that minimizing is relevant with disease or disease cause owing to the appearance of primary disease or disease.The order of severity that the employed phrase of this paper " avoids catching " and refers to the occurrence rate that reduces disease symptoms, reduces disease and/or reduce disease.The protection patient can refer to that it stoped the ability of disease appearance and/or healing or the symptom that palliates a disease, sign or reason when compositions of the present invention gave the patient.Therefore, make the patient avoid catching and comprise prophylactic generation (prophylactic treatment) and the ill patient's (therapeutic treatment) of treatment.Beneficial effect is easy to be assessed by the treatment patient's of those skilled in the art and/or process specialized training clinician.Term " disease " refers to that mammal departs from from any of normal health, comprises the state when disease symptoms exists, and departs from (as infection, gene mutation, genetic defect etc.) and occurred but disease that symptom is not confirmed as yet.

Therefore, method of the present invention comprises the purposes of plurality of reagents (being regulating compound), these reagent reduce airway hyperreactivity and/or the airway inflammation of animal thus by directly the albumen of the complement bypass selectivity that works being suppressed one or more proteic expression and/or biologic activity of complement bypass.Being used for reagent of the present invention comprises albumen for example, nucleic acid molecules, antibody and is the chemical compound (being medicine) of the product of reasoning drug design.These reagent refer generally to inhibitor in this article.According to the present invention, inhibitor is any inhibition (directly suppressing or the competitive inhibition) expression of albumen (as the albumen of complement bypass) and/or the reagent of biologic activity, comprises the reagent that the B factor, the D factor or properdin are worked.In one embodiment of the invention, the proteic reduction (promptly reduce, reduce, suppress) that is suppressed at the proteic biologic activity that is defined as any measurable (detectable) complement bypass herein of complement bypass or complement bypass.Proteic biologic activity or biological action refer to any function that (being proteic natural physiological environment) or external (promptly under laboratory condition) detected or observation post represents or carries out in the albuminous body of natural existence form.For example, the biologic activity of the B factor includes but not limited in conjunction with activated C3, lytic immunity complex, Bcell growth factor activity and monocyte activation.According to the present invention, proteic biologic activity can be suppressed by the ability that directly stops or suppress (reduce, reduce) protein binding and/or activate another kind of albumen (as C3), suppresses this thus in conjunction with caused downstream events.Preferably, the biologic activity of complement bypass is suppressed by at least a proteic reagent that suppresses in this approach, this reagent include but not limited to protein binding and/or activate that another kind of proteic ability is suppressed or the mode that stops in conjunction with at least a albumen in this approach or with this approach in the reagent of protein competition.This reagent includes but not limited to proteic antagonist, antibody (comprising its Fab), other antigen-binding polypeptides and micromolecule (as synthetic compound or medicine).

The antagonist that to be used for a kind of reagent of the present invention be complement bypass comprises the proteic antagonist in this approach.According to the present invention, " antagonist " refers to suppress any chemical compound that (as antagonism, reduction, minimizing, blocking-up, reverse or change) specifies proteic effect.More specifically, antagonist can be with respect to specifying proteic active mode to work, therefore the proteic biologic activity of this appointments with antagonism (as antagonism, reverse, in contrast to) mode of the proteic natural action of appointment is lowered or blocks.The product that antagonist can include but not limited to antibody or its Fab, albumen, peptide, nucleic acid (comprising ribozyme and antisense strand) or the medicine/chemical compound of antagonistic effect/peptide design is provided or select.For example, the present invention includes any antagonist of native protein, the B factor, the D factor or properdin, comprise antibody antagonist, albumen/peptide antagonists, nucleic acid antagonist or micromolecule antagonist (as micromolecular inhibitor).

In an embodiment preferred of the present invention, the reagent that is used to suppress complement bypass is antibody or its Fab.Equally, antigen-binding polypeptides also is particularly preferred among the present invention.On the one hand, the albumen of antibody selective binding complement bypass, (generally, i.e. natural endowment they interact down or under the physiological condition) is suppressed or stops so that this albumen and another kind of proteic the combination.On the other hand, this albumen of antibody selective binding is to suppress or to stop this albumen to activate another kind of albumen (generally interacting, even if this albumen combines with other protein part).Particularly preferred antibody and the Fab thereof that is used for selectivity inhibition complement pathway described (B factor antibody, particularly the mAb1379 antibody of this paper detailed description as described herein) hereinbefore in detail.

Preferably, be used for antibody of the present invention or its Fab in conjunction with the albumen that is selected from the B factor, the D factor or properdin.Most preferably, the present invention includes antibody or its Fab in conjunction with the B factor.The selective binding B factor of the present invention and the antibody (and Fab) that suppresses complement bypass are described in detail in this article and are illustrated.In one embodiment, antibody or its Fab are combined in animal species, particularly conservative this proteic conservative mating surface or epi-position (i.e. this antibody and this albumen cross reaction from two or more different mammalian species) in mammalian species.Particularly, the present invention includes in conjunction with proteic antibody from the complement bypass at least two kinds, preferred several different mammalian species (including but not limited to people, primates, mice, rat, pig, horse and rabbit except that the people).

The invention still further relates to design and come selective binding and neutralize or suppress proteic non-antibody polypeptide of the present invention, refer to antigen binding partners or antigen-binding polypeptides sometimes.The example of design with this polypeptide of aforementioned ligand specificity provides (Proc.Natl.Acad.Sci.96:1898-1903,1999) by people such as Beste, includes this paper in full in by quoting.

Except antibody, its Fab and antigen-binding polypeptides, the present invention also comprises proteic other reagent that suppresses complement bypass.These reagent for example comprise chemical compound, the natural product of the product of reasoning drug design and the chemical compound with the clear and definite regulation and control character of part.Regulation and control reagent comprises specifies proteic antagonist, can be chemical compound, the chemical compound based on carbohydrate, the chemical compound based on lipid, the chemical compound based on nucleic acid, natural organic compound, the synthetic organic compound of deriving, antibody or its fragment based on albumen.In one embodiment, this regulation and control reagent of the present invention comprises medicament, and these medicaments comprise one or more the proteic generations of regulating complement bypass and/or peptide, oligonucleotide, carbohydrate and/or the synthetic organic molecule of function.This reagent can obtain from the library of for example molecular diversity method (can make up the combination of the correlation technique of the bigger molecular library with Chemical Diversity fast), native compound or synthetic compound, particularly from chemical library or combinatorial library (but be sequence or vary in size have the library of compounds of identical construction unit) obtain, or obtain by the reasoning drug design.Referring to for example Maulik et al., 1997, MolecularBiotechnology:Therapeutic Applications and Strategies, Wiley-Liss, Inc. includes this paper in full by quoting.

In the molecular diversity method, use biological method, Enzymology method and/or chemical method, from for example synthesizing bigger library of compounds peptide, oligonucleotide, carbohydrate and/or the synthetic organic molecule.Key parameter in the conducting molecule multiformity method comprises subunit multiformity, molecular size and library multiformity.The general purpose that screens this library is to utilize continuous use combination selection to obtain the high-affinity part at required target material, then by method for designing or directed method for designing at random with the main molecules optimization.The method of molecular diversity is described in detail by people such as Maulik (above).

In the reasoning drug design method, the three dimensional structure of regulating compound can pass through for example nuclear magnetic resonance, NMR (NMR) or X-ray crystallography analysis.By for example computer model, use the structure of this three dimensional structure prediction such as possible chemical compound of possible adjusting control agent then.The structure of predicted chemical compound can be used for the main compound optimization that obtains by the molecular diversity method for example.In addition, predicted compound structure can pass through for example chemosynthesis, recombinant DNA technology or by producing from natural origin (as plant, animal, antibacterial and fungus) separation simulation epi-position.

People such as Maulik (1997, above) other the whole bag of tricks based on the drug design of structure is disclosed.People such as Maulik disclose for example method of orientation design (wherein user instructs the process of formation from the novel molecular in the segmental fragment library of appropriate selection), (wherein user use genetic algorithm or other algorithm random mutation fragment and the combination thereof of design at random, use the fit (fitness) of choice criteria simultaneously with the assessment candidate ligand) and based on the method for screen cloth (wherein user has calculated the interaction energy between three-dimensional receptor structure and the little fragment probe, then favourable probe site is linked together).

The isolated nucleic acid molecule that can be used as the proteic reagent that suppresses complement bypass is antisense nucleic acid molecule, ribozyme or siRNA.The employed antisense nucleic acid molecule of this paper be defined as by under stringent condition with the gene recombination of encoding proteins to reduce the isolated nucleic acid molecule of protein expression.The gene of this nucleic acid molecules and encoding proteins is closely similar, and this molecular energy is hybridized with the gene of coding native protein or coding strand or the complementary strand RNA of RNA under the height stringent condition.It is a kind of method that RNA disturbs (RNAi), and by this, the short interfering rna in double-stranded RNA and the mammlian system (siRNA) is used to suppress or the expression of reticent complementary gene.SiRNA in the target cell is unwind, and relevant with the inductive reticent complex of RNA (RISC), and RISC is entered the complementary mRNA sequence with siRNA by guidance subsequently, and RISC has sheared mRNA by this.Ribozyme is for by partly working in conjunction with target RNA and by shearing the RNA section that phosphodiester backbone makes its inactivation at specific shearing site.

Gene comprise the production of controlling gene encoding proteins control region (such as but not limited to transcribe, translation or post-translational control district) and coding region self.The gene of the multiple protein (comprising the B factor, the D factor or properdin) of coding complement bypass has been identified and has been known in the art.Isolated nucleic acid molecule can comprise the derivant of DNA, RNA or DNA or RNA for take out the nucleic acid molecules of (promptly being subjected to the people for controlling) from its natural surroundings.Therefore, " separation " do not reflect the degree that nucleic acid molecules is purified.Isolated nucleic acid molecule of the present invention can separate or use recombinant DNA technology (as polymerase chain reaction (PCR) amplification, clone) or chemosynthesis production from natural origin.

The employed hybridization conditions of this paper refers to that nucleic acid molecules is used to identify the standard hybridization conditions of similar nucleic acid molecules.For example, people such as Sambrook (Sambrook et al.Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Labs Press, 1989) disclose this standard hybridization conditions.This publication of people such as Sambrook (as preceding) is included this paper (specifically referring to the 9.31-9.62 page or leaf) in full by quoting.In addition, for example people such as Meinkoth (Meinkoth et al., 1984, Anal.Biochem.138,267-284) also disclose calculate suitable hybridization and wash conditions formula to obtain the hybridization of nucleotide mispairing in various degree.This publication of people such as Meinkoth (as preceding) is included this paper in full by quoting.

Ischemia reperfusion injury

Use anti-B factor antibody for example described herein or its Fab that another embodiment of the present invention relates to the inventor suppress the B factor and suppress this discovery of ischemia reperfusion injury.This discovery confirms in kidney ischemia reperfusion injury model.Therefore, method and composition of the present invention with ischemia reperfusion injury and an aspect of of the present present invention---also have treatment effectiveness in the relevant disease of kidney ischemia reperfusion injury.Use the ischemia reperfusion injury of other type that this method can prevent or reduce, include but not limited to the ischemia-reperfusion of ischemia reperfusion injury, internal organs such as lung, liver or small intestinal of heart ischemia-reperfusion injury, central nervous system's ischemia-reperfusion, extremity or finger (toe) or the ischemia reperfusion injury of any transplant organ or tissue.

Method of the present invention comprises the following steps: that selectivity suppresses to suffer from ischemia-reperfusion or the interior complement bypass of animal of suffering from this disease risk is arranged.Preferably, the symptom of at least one damage that causes owing to ischemia-reperfusion or type are prevented or are suppressed.Ischemia reperfusion injury can improve cell and organize the generation or the oxidation of the deleterious chemical compound of various possibilities that is produced, and this process can cause the oxidative damage or the death of cell and tissue.For example, the kidney ischemia reperfusion injury can cause the Histological injury of kidney, comprises that with acute tubular necrosis (acute tubularnecrosis) be the tubular injury and the variation of feature.Caused renal function obstacle makes usually by the secreted accumulation such as the nitrogenous waste of serum urea nitrogen (SUN) of kidney.Ischemia-reperfusion also can cause the damage such as the far-end organ of lung.This method is preferably utilized the novel B factor antibody of the present invention of above-detailed, and when it being suffered from ischemia-reperfusion or the animal that suffers from this disease risk is arranged, its prevents, reduces or suppress at least one because the damage symptom that ischemia-reperfusion causes.Any B factor antibody of the present invention described herein, its Fab or the antigen-binding polypeptides with similar binding specificity can be used in this embodiment of the present invention.This paper provides in the whole bag of tricks of the present invention about preferred dose, route of administration, has comprised the description of this antibody and relevant combination of agents thing and preparation and they are included in this embodiment.

It should be noted that this embodiment of the present invention is particularly related to the treatment of ischemia reperfusion injury, therefore, do not need to cause that the associated conditions of ischemia reperfusion injury or inducement are significantly reduced or " healing ".Method of the present invention can prevention in full force and effect or reduction infringement or the damage relevant with ischemia-reperfusion, perhaps improves or alleviate at least one symptom of this damage.Therefore, give reagent described herein or preparation and can be used for prevention or suppress ischemia reperfusion injury, although do not need to prevent fully this damage, preferred patient can obtain at least a treatment effectiveness from the use of this reagent or preparation.

The preparation relevant, compositions and method with embodiment of the present invention

Another embodiment of the present invention also comprises and contains the complement bypass inhibitor, the preparation of complement bypass selective depressant particularly as described above or compositions.Said preparation or compositions can be used for using in any method described herein and with any reagent described herein (novel B factor antibody as described herein).In one embodiment, said composition can be used for reducing or preventing the airway hyperreactivity of animal.In another embodiment, said composition can be used for reducing or preventing the ischemia reperfusion injury of animal.In another embodiment, said composition suppresses complement bypass by selectivity and is used for the treatment of or prevents disease or disease.Said preparation comprises: (a) inhibitor of complement bypass as described above; (b) pharmaceutically useful carrier.

In one embodiment, said preparation or compositions can comprise one or more other reagent, as are applicable to reduce to suffer from airway hyperreactivity, particularly suffer from the relevant airway hyperreactivity of inflammation or the antibiotic medicine of the inflammation of the animal that suffers from this disease risk is arranged.The antibiotic medicine can be any antibiotic medicine that is applicable to the inflammation that reduces the patient suffer from the inflammatory condition relevant with airway hyperreactivity, includes but not limited to corticosteroid (oral, suck and inject), beta-2-agonists (long-acting or fugitive), leukotriene instrumentality (inhibitor or receptor antagonist), cytokine or cytokine receptor antagonist, anti-IgE, phosphodiesterase inhibitor, sodium cromoglicate, Nedocromil (nedocrimal), theophylline and T cell function inhibitor.The particularly preferred antibiotic medicine that is used for this preparation comprises corticosteroid, leukotriene instrumentality and cytokine or cytokine receptor antagonist.

In another embodiment, said preparation or compositions can comprise one or more other reagent, as are applicable to other reagent of the ischemia reperfusion injury of prevention or reduction animal.This reagent includes but not limited to the inhibitor of antibiotic medicine or oxidation and free radical damage.

In another embodiment, said preparation or compositions can comprise one or more other reagent, as are applicable to that treatment activates relevant another kind of disease or other reagent of disease with complement bypass.

According to the present invention, " pharmaceutically suitable carrier " comprises and is applicable to pharmaceutically acceptable excipient and/or the pharmaceutically useful delivery vectors that preparation or compositions is given intravital appropriate site.The preferred complement bypass of intravital appropriate site can repressed any site.In a preferred embodiment, when the patient suffered from airway hyperreactivity and/or airway inflammation or this disease risk of suffering from is arranged, intravital appropriate site was preferably in lung tissue or air flue.Intravital other preferred sites comprises other tissue or the organ of the infection center that is positioned at the disease relevant with complement bypass.In another embodiment preferred, intravital appropriate site is any site that ischemia reperfusion injury occurs, as in heart or pulmonary system, central nervous system, extremity or finger (toe), internal organs (as lung, liver or small intestinal) or at any transplant organ or in organizing.Preferred pharmaceutically suitable carrier is for being kept for the reagent of preparation of the present invention in the following manner: in case reagent arrives patient's target site, just reagent can act on its target material (as being the albumen of the component of complement bypass), preferably the patient is produced treatment effectiveness.

Suitable excipient of the present invention comprise transportation or assistant conveyance but not specificity make the excipient or the preparation (formulary) of compositions targeted cells or tissue (also referring to non-targeting vector in this article).The example of pharmaceutically useful excipient include but not limited to water, phosphate-buffered saline, Ringer solution, glucose solution, contain serum solution, Hank solution, other equilibrated aqueous solution, oils, esters and ethylene glycol on the physiology.Aqueous carrier can contain the suitable auxiliary substance that is required near experimenter's physiological condition, for example by improving chemical stability and isotonia.Suitable auxiliary substance for example comprises that sodium acetate, sodium chloride, sodium lactate, potassium chloride, calcium chloride and other are used to produce the material of phosphate buffer, Tris buffer and bicarbonate buffer.Auxiliary substance also can comprise antiseptic, as thimerosal, metacresol or orthoresol, formalin and benzyl alcohol (benzol alcohol).Can sterilize to preparation of the present invention by conventional method and/or lyophilization.

A kind of pharmaceutically useful carrier comprises the controlled release preparation that compositions of the present invention slowly can be discharged into animal.The employed controlled release preparation of this paper is included in the reagent of the present invention in the controlled release carrier.Suitable controlled release carrier includes but not limited to biocompatible polymer, other polymeric matrices, capsule, microcapsule, microgranule, inject preparation, osmotic pumps, dispersal device, liposome, lipid ball (liposphere) and percutaneous send delivery system.Other suitable carriers comprises and can wait to send the reagent of the half-life of passing reagent or include in this reagent in conjunction with prolongation.This carrier can comprise any suitable protein carrier, even comprises and send the fusion that prolongs the proteic half-life when passing section in vivo.Preamble has been described suitable delivery vectors, and they include but not limited to liposome, viral vector or comprise other delivery vectors of ribozyme.The natural delivery vectors that contains lipid comprises cell and cell membrane.The artificial delivery vectors that contains lipid comprises liposome and micelle.As mentioned above, can modify the specific site of delivery vectors of the present invention, thus targeting and the inhibitor that utilizes this site with the targeting patient.Suitable modification comprises the chemicals of the lipid part of controlling delivery vectors and/or import the targeting agent of the selectively targeted delivery vectors of energy to preferred sites (for example a kind of preferred cell type) in carrier.Other suitable delivery vectors comprises gold grain, poly-L-Lysine/DNA-molecular conjugate and artificial chromosome.

In one embodiment, the reagent that is used for this method is passed or nose send and passs, particularly is applicable to that aerosol send the preparation of passing (also referring to aerosol formulations in this article) to give to be applicable to that pulmonary send.This send the approach of passing to be specially adapted to prevent or suppress patient's AHR and/or the method for airway inflammation in, but also can be used for and it need be sent in other diseases that are delivered in lung or the air flue.In addition, these preparations are specially adapted to send and pass antibody.Said preparation generally comprises carrier, preferred pharmaceutically suitable carrier.Of the present inventionly be specially adapted to that aerosol send the carrier of passing to include but not limited to dehydrated alcohol, can disperse dry powder, compact capsule (as microcapsule or microgranule), liposome, injectable excipient and spray.Described and be used to send the dehydrated alcohol of passing albumen and peptide, for example referring to Choi et al., 2001, PNAS USA 98 (20): 11103-11107.Describe in detail and be applicable to that aerosol send the disperseed dry powder of passing reagent, for example referring to U.S. Patent No. 6,165,463, include in full this paper (also can be referring to from Inhale Therapeutic Systems, Inc., the product of nowNektar and Quadrant Technology) in by quoting.The suitable liposome that is used for spray comprises any liposome, send enough little any liposome of passing by aerosol in method of the present invention.Microcapsule and microgranule are known in the art.For example, AlliancePharmaceutical Corporation has the particulate technology technology that is called PulmoSphere, and this is by proprietary spraying-drying means preparation, and it is porous to be designed to hollow.The product of Ventolin is by being suspended in based on micronization albuterol (free alkali) granulometric composition in the mixture of the propellant of CFC.Proventil HFA contains micronization albuterol sulfate (albuterolsulfate) and the small amount of ethanol cosolvent oleic acid surfactant with steady dissolution.Including medicine in liposome send with regard to aerosol and has several advantages with regard to passing.Because liposome is soluble relatively,, improved drug effect so the retention time of some drugs in lung can be extended.Liposome is mainly taken in by phagocyte, and this makes liposome be specially adapted to sending of some drugs and passs.Spray agent is described in an embodiment.Send the device of passing aerosol formulations to include but not limited to that (metered solutiondevice MSI) and ultrasonic inhaler, and comprises the device of nebulizer and inhaler for pressure measurement inhaler (MDI), Diskus (DPI), metering solution device.Plurality of reagents can be used for sending in the preparation of passing by this device, send the suspension auxiliary agent passed and solubilizing agent (as the M-PEGS of lact-acid oligomer (oligolactic acid), acyl group-amic acid (acyl-amideacid) and single functionalization (mono-functionalized), referring to Mckenzieand Oliver as being used in particular for albumen; 2000; Formulating Therapeutic Proteins and Peptides inPressurized Metered Dosed Inhalers For Pulmonary Delivery; 3MHealth Care Ltd., Morley Street, Loughborough, Leicesteshire LE111EP, UK).

Pharmaceutically suitable carrier of energy targeting refers to " targeting delivery vectors " in this article.Targeting delivery vectors of the present invention can send passs the preparation that the comprises inhibitor target site to the patient." target site " refers to send the site in the patient who passs the treatment preparation.For example, target site can be for by antibody of the present invention or by using liposome, viral vector or other delivery vectors (comprising ribozyme) direct injection or sending any cell or tissue of passing institute's targeting.Can modify delivery vectors of the present invention or antibody specific site, thus at this site targeting with utilize specific compound, antibody, albumen or nucleic acid molecules with the targeting animal.Suitable modification comprises the chemicals of the lipid part of controlling delivery vectors and/or import the chemical compound of the selectively targeted delivery vectors of energy to preferred sites (for example a kind of preferred cell or tissue type) in carrier.Particularly, targeting refers to make delivery vectors be attached to specific cells by the interaction of molecules of carrying intravital chemical compound and cell surface.Suitable target compound comprises that energy selectivity (being specificity) is combined in the part of the another kind of molecule of specific site.The example of this part comprises antibody, antigen, receptor and receptors ligand.Useful especially example comprises any part relevant with complement pathway (as CR2, C3, C3d, C3dg, iC3b, C3b) or any part relevant with cell type, types of organization or the site of pending animal.Control the outer or born of the same parents' internal target tropism of born of the same parents of chemicals scalable delivery vectors of the lipid part of delivery vectors.For example, a kind of chemical substance can be added on the lipid formulations of liposome, with the electric charge of the double-layer of lipoid that changes this liposome, liposome merges mutually with the specific cells with specific charge feature like this.

A kind of delivery vectors that is used for various route of administration and reagent is a liposome.The lipid physical ability keeps stable and send the preferred sites that is delivered to animal with the nucleic acid molecules described in the present invention or even albumen or antibody in the sufficiently long time in animal.Liposome according to the present invention comprises the lipid compounds that the nucleic acid molecules of describing among the present invention can be sent on the specific or selected site that is delivered to animal.Liposome of the present invention comprises merging mutually so that nucleic acid molecules is sent with the plasma membrane of target cell and is delivered to intracellular lipid composition.Be used for suitable liposome of the present invention and comprise any liposome.Preferred liposome of the present invention comprises the liposome that for example is generally used in the known gene delivery method of those skilled in the art.Preferred liposome comprises the liposome with polycationic lipid compositions and/or has the liposome of the cholesterol skeleton of puting together with Polyethylene Glycol.Use can be united liposome and nucleic acid molecules of the present invention or inhibitor in this area standardized means.

Another kind of delivery vectors comprises viral vector.Viral vector comprises the isolated nucleic acid molecule that is used for method of the present invention, and wherein nucleic acid molecules is packaged in the virus coat, and this allows DNA to enter into cell.Can use multiple viral vector, include but not limited to viral vector based on α virus, poxvirus, adenovirus, herpesvirus, slow virus, adeno-associated virus and retrovirus.

According to the present invention, those skilled in the art can determine acceptable scheme, to give reagent, compositions or preparation, the effective dose that comprises the approach of giving and give the reagent of animal.Reagent of the present invention can be in vivo or external giving.That the approach that gives in the suitable body can include but not limited to is oral, in the nose, suction, part, trachea, percutaneous, rectum and parenteral route.That preferred parenteral route can include but not limited to is subcutaneous, intradermal, intravenous, intramuscular and intraperitoneal approach.Preferred local approach comprises the skin that is administered to animal by aerosol suction (promptly spraying) or local surfaces.Preferably, reagent by in the nose, suction, trachea, part or whole body approach (as intraperitoneal, intravenous route) administration.External finger carries out the part dosing step in patient's outside.The approach that preferably gives antibody comprises parenteral route and aerosol/nose/inhalation route.

Can use this area standardized means to carry out intravenous, intraperitoneal and intramuscular administration.Use this area standardized means to carry out aerosol (suctions) and send and pass (referring to for example Stribling etal., Proc.Natl. Acad.Sci.USA 189:11277-11281 1992, includes in herein by quoting full text).Be applicable to that aerosol send the carrier of passing to describe hereinbefore.The device that is used to send aerosol formulations includes but not limited to pressure measurement inhaler (MDI), Diskus (DPI) and metering solution device (MSI), and comprises the device of nebulizer and inhaler.Oral area send to pass to unite by the carrier that therapeutic combination of the present invention and the digestive enzyme in the digestive tract that can resist animal are degraded and carries out.The example of this carrier comprises plastic capsule or tablet, as those plastic capsule known in the art or tablet.The direct injection technology is specially adapted to recombinant nucleic acid molecules is given by the come-at-able cell or tissue of performing the operation, and particularly is administered on the surface of health or the cell or tissue of near surface.The compositions topical administration is referred to the target cell zone compositions is expelled to several centimeters apart from target cell or tissue, preferably apart from its several millimeters places.

Having shown that various medication disclosed herein and delivery vectors can effectively send nucleic acid molecules is delivered to target cell or tissue, thus nucleic acid molecules transfectional cell and being expressed.In many researchs, the success of heterologous gene is sent and is passed and be expressed in the preferred cell type and/or use preferred delivery vectors of the present invention and route of administration realizes.For example, using liposome to send passs, licensed to people's such as Dow U.S. Patent No. 5 on January 6th, 1998,705,151 show in the cationic-liposome delivery vectors successful terrain angular vein send the nucleic acid molecules of passing the coding superantigen and the nucleic acid molecules of the Codocyte factor, and encoding proteins is in the tissue of animal, especially express in the lung tissue whereby.People such as Liu (1997) illustrate vein and send and pass preferably targeting lung tissue and effectively intragentic transfer of control agent and the expression of the cationic-liposome that contains cholesterol that contains gene.Can finish sending of multiple nucleotide sequence by the viral vector that gives nucleic acid sequence encoding passs.

The single dose that preferably is used for the reagent (comprising albumen, micromolecule and antibody) of any method described herein contains between about 0.01 microgram * kilogram ^-1To about 10 milligrams * kilogram ^-1The weight of animals.The single dose of more preferred reagent comprises between about 1 microgram * kilogram ^-1To about 10 milligrams * kilogram ^-1The weight of animals.The single dose of more preferred reagent comprises between about 5 microgram * kilograms ^-1To about 7 milligrams * kilogram ^-1The weight of animals.The single dose of more preferred reagent comprises between about 10 microgram * kilograms ^-1To about 5 milligrams * kilogram ^-1The weight of animals.If reagent send by aerosol and passs, the single dose of particularly preferred reagent comprises between about 0.1 milligram * kilogram ^-1To about 5 milligrams * kilogram ^-1The weight of animals.If reagent is sent outside by the intestines and stomach and passed, the single dose of another particularly preferred reagent comprises between about 0.1 microgram * kilogram ^-1To about 10 microgram * kilograms ^-1The weight of animals.

In one embodiment, suitable nucleic acid of the present invention: the single dose of liposome complex is about 0.1 μ g gives complex to about 100 μ g/kg a weight in patients.In another embodiment, suitable single dose is that about 1 μ g is to about 10 μ g/kg body weight.In another embodiment, suitable nucleic acid: the single dose of lipid complex is at least about 0.1 μ g nucleic acid, more preferably at least about 1 μ g nucleic acid, more preferably at least about 10 μ g nucleic acid, more preferably at least about 50 μ g nucleic acid, more preferably at least about 100 μ g nucleic acid.

In one embodiment, be used for any method described herein reagent of the present invention suitable dose for do not give this reagent and compare and effectively suppress the expression or the active dosage of at least a albumen (as the B factor, the D factor or properdin) of complement bypass described herein.The method that detects proteic expression or biologic activity has above been described.In another embodiment, the suitable dose of reagent of the present invention is for can significantly suppressing the dosage of complement bypass of the present invention.Use technology known in the art/analysis can detect complement activation and inhibition thereof.For example, can carry out C3 lithosomic body outer analysis on the described in an embodiment zymosan A granule.Also can test this reagent and suppress the not erythrocytic ability of sensitization of human serum cracking.Based on these analyses, obtain in the body in dosage those skilled in the art limit of power by in vitro results extrapolation.

Well knownly in the people, use conventional method to carry out aerosol to send and pass, even use inhaler, 10% send and pass solution and enter into the air flue depths of generally only having an appointment.If aerosolization is sent and passed is by direct suction, can suppose that just dosage is by about 10% of dosage that spray method gives.At last, use analogy method (allometric scaling), those skilled in the art is easy to mice is converted into human dosage with dosage.In fact, mice to transform to people's dosage be based on the clearance rate of chemical compound and the body surface of mice." not observing the ill effect level " (NOEL) 1/12 concentration with acquisition human dosage of value is got in the conversion of mg/kg.Removing between this calculating hypothesis mice and the people is identical, and antagonist it is believed that true like this really.

Therefore, the single dose of preferred antibody comprises between about 1ng * kilogram ^-1To being less than 1mg * kilogram approximately ^-1The weight of animals.The single dose of preferred antibody comprises between about 20ng * kilogram ^-1To about 600 μ g * kilogram ^-1The weight of animals.The single dose of more preferred antibody, particularly when antibody preparation sending by nebulization when passing, comprise between about 20ng * kilogram ^-1To about 600 μ g * kilogram ^-1The weight of animals, more preferably, between about 20ng * kilogram ^-1To about 500 μ g * kilogram ^-1, more preferably, between about 20ng * kilogram ^-1To about 400 μ g * kilogram ^-1, more preferably, between about 20 ng * kilograms ^-1To about 300 μ g * kilogram ^-1, more preferably, between about 20ng * kilogram ^-1To about 200 μ g * kilogram ^-1, more preferably, between about 20ng * kilogram ^-1To about 100 μ g * kilogram ^-1, more preferably, between about 20ng * kilogram ^-1To about 50 μ g * kilogram ^-1The weight of animals.

The single dose of another preferred antibody particularly send when passing by nebulization at antibody preparation, comprises between about 200ng * kilogram ^-1To about 600 μ g * kilogram ^-1The weight of animals, more preferably, between about 200ng * kilogram ^-1To about 500 μ g * kilogram ^-1, more preferably, between about 200ng * kilogram ^-1To about 400 μ g * kilogram ^-1, more preferably, between about 200ng * kilogram ^-1To about 300 μ g * kilogram ^-1, more preferably, between about 200ng * kilogram ^-1To about 200 μ g * kilogram ^-1, more preferably, between about 200ng * kilogram ^-1To about 100 μ g * kilogram ^-1, more preferably, between about 200ng * kilogram ^-1To about 50 μ g * kilogram ^-1The weight of animals.

The single dose of another preferred antibody particularly send when passing by direct inhalation from inhaler at antibody preparation, comprises between about 2ng * kilogram ^-1To about 100 μ g * kilogram ^-1The weight of animals, more preferably, between about 2ng * kilogram ^-1To about 50 μ g * kilogram ^-1, more preferably, between about 2ng * kilogram ^-1To about 10 μ g * kilogram ^-1, more preferably, between about 2ng * kilogram ^-1To about 5 μ g * kilogram ^-1, more preferably, between about 2ng * kilogram ^-1To about 1 μ g * kilogram ^-1, more preferably, between about 2ng * kilogram ^-1To about 0.5 μ g * kilogram ^-1, more preferably, between about 2ng * kilogram ^-1To about 0.25 μ g * kilogram ^-1, more preferably, between about 2ng * kilogram ^-1To about 0.1 μ g * kilogram ^-1The weight of animals.

In another embodiment, antibody gives with the dosage that is less than about 500 μ g antibody/ml of formulation, preferably be less than about 250 μ g antibody/ml of formulation, more preferably, be less than about 100 μ g antibody/ml of formulation, more preferably, be less than about 50 μ g antibody/ml of formulation, more preferably, be less than about 40 μ g antibody/ml of formulation, more preferably, be less than about 30 μ g antibody/ml of formulation, more preferably, be less than about 20 μ g antibody/ml of formulation, more preferably, be less than about 10 μ g antibody/ml of formulation, more preferably, arrive about 10 μ g antibody/ml of formulation between about 5 μ g antibody.

Specifically about reducing or prevention airway hyperreactivity and/or airway inflammation or the disease of being correlated with therewith or the method for disease, when the single dose of the suitable inhibitor that gives animal refers to that single or multiple gives in one appropriate period, can reduce or prevent airway hyperreactivity and/or the airway inflammation of animal, or reduce the dosage of at least a other symptom that disease to be treated (as asthma) is arranged.When the patient suffers from AHR or has when suffering from this disease risk, the single dose of suitable reagent comprises that the exciting agent dosage with AHR increases to twice or improves the dosage of the static respiratory function of animal.

The method according to this invention gives effective dose that animal suppresses the reagent of AHR and comprises and can reduce airway hyperreactivity (AHR) or airway inflammation does not have deleterious amount to animal simultaneously.The deleterious amount of animal is comprised any structure of animal or amount of functional lesion (promptly poisonous) of causing.

In one embodiment, the AHR inhibitor makes that suffer from AHR or have the animal that suffers from this disease risk to avoid infecting the effectiveness of AHR can be by double measurement amount.For example, if the PC of animal _{20 acetonyl choline}FEV ₁Being 1mg/ml before giving reagent, and being 2mg/mlMch after giving reagent, is significant by the ability (promptly reducing or prevention) of specifying reagent to make animal avoid infecting AHR so.Equally, if the PC of animal _{20 acetonyl choline}FEV ₁Be 2mg/ml before giving reagent, and be 4mg/ml Mch after giving reagent, then reagent is considered to effective.The effective dose of preferred reagent comprises can be through the PC of the animal of this agent treated _{20 acetonyl choline}FEV ₁PC to the intact animal _{20 acetonyl choline}FEV ₁Direction increases to the amount of about double strength.As mentioned above, the intact animal refers to knownly not suffer from unusual AHR or to its insensitive animal.Tested animal refers under a cloudly suffer from unusual AHR or to its responsive animal.

In one embodiment of the invention, in suffering from the animal of AHR, give animal reagent effective dose for give reagent before compare the amount of the AHR of remarkable reduction animal.In another embodiment, give animal reagent effective dose for have the level of air flue AHR that the AHR related inflammation do not give the animal population of this reagent and compare the amount of the AHR of remarkable reduction animal.Reagent preferably can reduce the AHR of animal, or even shows effect back (being acute AHR outbreak back) when giving reagent at the physical symptom of AHR.More preferably, AHR no longer can detected amount in the patient for the symptom with AHR is reduced to for the effective dose of reagent.In another embodiment, the effective dose of reagent is to be exposed to when giving reagent before AHR such as anaphylactogen excites stimulus object the patient, can prevent or significantly suppress the amount of AHR outbreak, and wherein this process-exposed is enough to induce AHR under the situation of not having this reagent.

In another embodiment, the effective dose of the reagent of method of the present invention comprises the PC that causes animal _{20 acetonyl choline}FEV ₁The amount that value improves, wherein before giving this reagent, the PC that animal is obtained when exciting with the acetonyl choline of first concentration _{20 acetonyl choline}FEV ₁Value with give this reagent after, the PC that animal is obtained when exciting with the acetonyl choline of first concentration of double amount _{20 acetonyl choline}FEV ₁Be worth identical.The preferred amount of reagent comprises the PC that causes animal _{20 acetonyl choline}FEV ₁The amount that value improves is wherein at the PC that gives to be obtained before this reagent _{20 acetonyl choline}FEV ₁Value arrives between about 8mg/ml acetonyl choline between about 0.01mg/ml, this and the PC that is obtained after giving this reagent _{20 acetonyl choline}FEV ₁Be worth identical, this moment should value between about 0.02mg/ml arrives about 16mg/ml acetonyl choline.

As mentioned above, reagent protection suffers from AHR or can give FEV before and after the reagent by measurement to the effectiveness of its responsive animal ₁Raising percentage ratio and/or FEV ₁/ FVC detects.In one embodiment, the effective dose of reagent comprises that the flow limitation that can reduce animal is so that the FEV of animal ₁/ FVC value is at least about 80% amount.In another embodiment, the effective dose of reagent comprises that the flow limitation that can reduce animal is so that the FEV of animal ₁It is about 5% that/FVC value improves at least, or at least about the amount of 100cc or PGFRG 10L/min.In another embodiment, the effective dose of reagent comprises the FEV that makes animal ₁Raising at least about 5%, more preferably improve about 6% to about 100%, more preferably improve about 7% to about 100%, more preferably improve about 8% expectation FEV to about 100% (or about 200ml) animal ₁Amount.In another embodiment, the effective dose of reagent comprises the FEV with animal ₁At least improve about 5%, preferably improve at least about 10%, more preferably improve at least about 25%, more preferably improve at least about 50%, more preferably improve about 75% amount at least.

The dosage number that those skilled in the art can determine to give the reagent of animal depends on the degree of airway hyperreactivity, be the basic disease of symptom and the reactivity that individual patients to be treated is arranged with this AHR.In addition, the clinicist can determine effectively to reduce animal AHR send the appropriate time of passing reagent.Preferably, being exposed to amount that the AHR that can effectively induce AHR excites stimulus object the patient send in preceding 48 hours and passs reagent, more preferably in the patient is exposed to preceding 36 hours of the amount that the AHR that can effectively induce AHR excites stimulus object, more preferably in 24 hours, more preferably in 12 hours, more preferably in 6 hours, 5 hours, 4 hours, 3 hours, 2 hours or 1 hour.In one embodiment, patient or clinicist recognize the patient be exposed to maybe will be exposed to AHR excite stimulus object, when particularly being exposed to the AHR that maybe will be exposed to the sensitization patient and having excited stimulus object (being anaphylactogen) (immediately) give this reagent.In another embodiment, at the first reagent that just gives when finding to occur AHR (being acute AHR outbreak), preferably at least in AHR symptom at least 1 hour appears in 2 hours, preferably occurring in the AHR symptom, more preferably at least 30 minutes, more preferably at least 10 minutes, more preferably at least 5 minutes.Above describe symptom and the measurement of AHR in detail or detected the method for this symptom.Preferably, carry out this and give, the sign that reduces up to AHR occurs, and carries out as required then, up to the transference cure of AHR.

Specifically about suppressing or prevent the method for ischemia reperfusion injury, the effective dose of reagent, the effective dose that particularly gives the B factor antibody of animal or its Fab (or antigen-binding polypeptides) significantly suppresses the amount of histology's infringement (comprising oxidative damage or cell death) of animal for to compare with not giving this reagent.With regard to the kidney ischemia reperfusion injury, give animal reagent effective dose for do not give this reagent and compare the rising of remarkable inhibition serum urea nitrogen or significantly reduce the amount of the Histological injury of animal kidney tissue.The single dose of the suitable inhibitor that gives animal can reduce or prevent the dosage of at least a symptom, type of impairment or caused infringement of the ischemia reperfusion injury of animal when giving one or many in one suitable period.Above describe the suitable dose of antibody in detail, comprised the suitable dose that is used for various route of administration.On the one hand, the effective dose of reagent that gives the inhibition ischemia reperfusion injury of animal comprises and can suppress by caused at least a symptom of ischemia reperfusion injury or infringement, nontoxic to animal simultaneously amount.

Any method of the present invention can be used for any animal of any animal, particularly mammals (being mammal) vertebrates guiding principle, includes but not limited to primates, Rodents, domestic animal and raises and train house pet.Preferred mammal of using method of the present invention to treat is the mankind.

Following examples are the property purpose presented for purpose of illustration, is not intended to limit scope of the present invention.

Embodiment

Embodiment 1

The following example has been described the production of the new inhibitor of complement bypass.

The inventor has made several hybridomies of producing in conjunction with the mouse monoclonal antibody of the mice B factor.In this research, the inventor sets about identifying the wherein a kind of ability that suppresses complement bypass in these antibody.The inventor has also checked this antibody in the fetal loss model of anti-phospholipid mediation.As (Girardi) as described in the forefathers, the mice of B factor disappearance avoids fetal loss in this model, and the inventor supposes that the external source inhibitor of alternative pathway is effectively to treat reagent in this disease model.

Method

The purification of the structure of the B factor-Ig fusion rotein and the mice B factor.Having made up the plasmid (Fig. 1) of two short consensus repeats (SCR) of B factor gene that coding connects hinge region, CH2 district and the CH3 district of mice IgG1 isotype.Select these SCR domains to be because they be employed fB-in these researchs/-part of the disappearance section of the B factor gene of mice.

The purification of the mice B factor.By affinity purification purification complement B factor from normal mouse serum.According to the description of manufacturer, by (Diasorin, Stillwater MN) are attached to CNBr-Activated Separose (Amersham, Arlington Heights, IL) preparation affinity column with the anti-people's BF of goat.By cardiac puncture the C57/B6J mice is taken a blood sample, and collect blood in the syringe that contains 50 μ l 500mM EDTA, activate to prevent alternative pathway.Blood at the centrifugal 15min of 2000rpm, is collected blood plasma.(PBS that contains EACA 50 mM, EDTA 10 mM, benzamidine 2 mM, pH7.4) 1: 1 diluting plasma use 0.22 μ m filter (GE Water Technologies) to filter then to use buffer then.Blood plasma is added affinity column, and the buffer with 10 times of column volumes washes this post then.With 5 M LiCl ₂The eluting B factor is also used the PBS dialyzed overnight.Electrophoresis is also used and is examined the purity (not providing data) that the dyeing of Ma Shi light blue detects the B factor on the 10%Tris-Glycine gel then.

The exploitation of the inhibition type monoclonal antibody of the targeting complement B factor.Finish the targeting disappearance of the mice B factor as (Matsumoto) as described in preceding going into.Mice with embryonic stem cell Sv129 cell strain prepares B factor disappearance hybridizes with the C57BL/6 mice before F1 colony propagation then.Mice with the emulsive reorganization of the incomplete Freund B factor-Ig fusion rotein 125 μ g immunity B factor disappearance carries out booster immunization four times, each three weeks at interval then.By using mice B factor bag, screen the mice of the inhibition type antibody that the anti-B factor occurs with this by analyzed in vitro (vide infra) the check mice serum of Enzyme Linked Immunoadsorbent Assay of culture plate (ELISA) and complement bypass inhibition.Injection in the end will be had at splenocyte of the mice of the strong immune response of the B factor and myeloma cell line from Colorado university monoclonal antibody center (University of Colorado Monoclonal Antibody Center) by evaluation and merge one day after.By limiting dilution assay clone candidate hybridoma, evaluation can suppress the active clone of alternative pathway.A kind of hybridoma A1379 is used to these experiments.With Protein-G Sepharose post (Pharmacia, Uppsala, Sweden) purification A1379 from tissue culture's supernatant.Use polymyxin (Sigma) that LPS is removed from the mAb of purification.According to the description of manufacturer use tachypleus amebocyte lysate measure (Limulus AmebocyteLysate Assay, BioWhittaker, Inc., Walkersville, MD) whether the LPS level of check mAb is lower than 1 EU/mg mAb.Electrophoresis is also used the purity of examining Ma Shi light blue chromoscopy mAb on the 10%Tris-Glycine gel then.

The elisa assay of anti-B factor antibody level

With Enzyme Linked Immunoadsorbent Assay (ELISA) check mice serum, screen the mice that immunity inoculation is produced immunne response at the purification B factor with this.Be cushioned liquid (15mMNa with being dissolved in bag ₂CO ₃, 35mM Na ₂HCO ₃) the purification B factor 125 ng bag (Costar, Corning is NY) and 4 ℃ of preservations of spending the night by 96 hole elisa plates.Wash this plate with 200 μ l PBS then.By (MO) 200 μ l are hatched these plates for Sigma-Aldrich, St.Louis, the blocking-up non-specific binding with the 5%BSA that is dissolved in PBS.Plate with the PBS 200 μ l washed twice that contain 0.1%Tween 20, was hatched 1 hour with dilute serum then.With 1: 100 diluted sample of the PBS that contains 0.1%Tween20 and 0.1%BSA, then this sample was carried out serial dilution seven times with 1: 1.Then with the plate washed twice and put together the peroxidase of goat anti-mouse IgG with 50 μ l (Cappel, Durham NC) are hatched.(contain 1: 1000 30%H with plate washing four times and with 100 μ l ABTS then ₂O ₂) (Sigma) hatch together, and (Biorad, Richmond CA) read light absorption value at 405 nm with the microtest plate reader.

The analysis that complement bypass suppresses.The serum that will have the anti-B factor antibody that can detect titre then screens with regard to the ability that it suppresses alternative pathway.This carries out C3 lithosomic body outer analysis by use and carries out (Quigg) on zymosan A granule (Sigma).The 50mg zymosan granule that will be dissolved among the 10ml 0.15M NaCl boiled 60 minutes, used the PBS washed twice then.By with 1 * 10 in the reactant mixture ⁷Zymosan granule and final concentration are respectively EGTA and the MgCl of 10mM and 5mM ₂Hybrid analysis serum.The serum that 10 microlitres are not controlled the C57/B6 mice is used as the adding of complement source.Carry out inhibition analysis with 0.0625 μ g to the titrating antibody purification of 8 μ g with the serum that is up to 70 μ l immune mouses (with the formation of screening inhibition type antibody) or with each reaction.Is 100 μ l final volume with PBS with the sample dilution, and hatches 30min at 37 ℃.The zymosan granule is with cold PBS, 1% hyclone washed twice, and (Capple, Durham NC) are hatched 1 hour on ice to the goat anti-mouse C3 that puts together with FITC then.With sample washed twice again, resuspended with 0.5ml PBS, 1% hyclone, analyze by flow cytometry then.Use following formula to calculate and suppress percentage ratio:

Also use the zymosan analysis it to be checked with regard to the ability of 1379 clones' Fab fragment inhibition alternative pathway.According to the description of manufacturer, by (ICN Biomedicals, Aurora OH) are hatched together and formed the Fab fragment with antibody purification and papain-agarose.Be applied to by antibody then and remove Fc fragment and indigested IgG on the G albumen post digestion.In effluent, collect the Fab fragment, use 0.1M glycine-hydrochloric acid (pH2.8) eluting Fc fragment and indigested IgG successively then.In the zymosan reaction, used 1 μ g Fab.Employed polyclone anti-mice C3 antibody and multiple species generation cross reaction in this analysis have been found.Therefore, this analysis can be used for checking the inhibitory action of 1379 clones to the alternative pathway of these species.As mentioned above this inhibition type antibody is carried out titration.

As another analysis to the ability of 1379 clone's inhibition complement bypasses, the inventor has checked this antibody to suppress the not ability of sensitization rabbit erythrocyte of human serum cracking.Rabbit whole blood and buffer are mixed at 1: 1, and this buffer is by containing 20.5g glucose, 8.0g (two hydrations) sodium citrate, 4.0g NaCl, 0.55g citric acid formation in 1 liter of distilled water.Then with the 5ml solution of red blood cells with contain 1.1%NaCl, 0.0025%5,5 diethyl barbital sodium (Na-5,5 diethylbarbiturate), pH7.35,8mM EGTA, 2mM MgCl ₂Solution 1: 9 mix.This mixture is hatched a few minutes for 37 ℃, then centrifugal 10 minutes of 4 ℃ of 1000 * g.This erythrocyte is washed three times again, be resuspended in the 40ml same solution then.The above-mentioned suspension of 50 μ l is added in human serum (5 to the 100 μ l) buffer solution, make that final volume is 150 μ l.Erythrocyte in the serum-free buffer is as negative control, adds erythrocyte in the 100 μ l distilled water as positive control (cracking fully).Sample is hatched 30 minutes at 37 ℃, and vibrate once in a while to keep cell suspension.Add the cold PBS cessation reaction of 1.5ml, then with sample centrifugal 5 minutes at 1000 * g.Use spectrophotometer (Biorad) to read the optical density value of each supernatant at 415nm.Find that 10 μ l serum make the complete cracking of erythrocyte.1379 clones' (1 μ g reacts to 12 μ g/) that use 10 these serum of μ l and concentration to increase then carry out same reaction.The active inhibition percentage rate of alternative pathway uses following formula to measure:

1379 clones' body giving drugs into nose is for kinetics.With the mice blood sampling, the antibody 1379 of intraperitoneal injection then (IP) or intravenous injection (IV) 1mg or 2mg dosage is cloned earlier.Select these dosage to be because they and the B factor etc. mole according to estimates.The B factor in the serum is approximately～200 μ g/ml (or～2.2 μ M, if the B factor is the albumen of 90kD).Be about 3ml because 1379 antibody are the intravascular volume of 150kD and adult mice, inject 1 mg (6.7 μ Mol) will produce～circulation composition of 2.2 μ M.Because antibody is two valencys, these moles injection is expected to more effectively cause the inhibition fully of alternative pathway.Behind the injection inhibitor, mice taken a blood sample in 1,2,6,24,48 and 96 hour.To be used for the activity of zymosan analysis then from the serum of these time points with the assessment alternative pathway.

The result

Formation at the inhibition type monoclonal antibody of the Ba of B factor part.Such as method part form monoclonal antibody the description at the mice B factor.Serum from immune mouse is used for analyzing whether have anti-B factor antibody (not providing data).Select a kind ofly to be called 1379 clone and to be used for further sign, this is owing to find this hybridoma and can grow fast that this antibody is the subclass (non-complement activation) of IgG1 and finds that its supernatant is effective inhibitor of complement bypass.To (not provide data) behind the antibody purification, in two activated analyzed in vitro of alternative pathway, checked this antibody (Fig. 2 A and 2B).When 3 μ g inhibitor being added in the reaction system that contains 10 μ l serum, use the zymosan analysis to find that this inhibitor has suppressed alternative pathway fully.The anti-B factor antibody and the B factor almost wait molar concentration (suppose that the B factor is 200 μ g/ml, molecular weight is 90,000 kD, is 0.022nMol, and 3 μ g molecular weight are that the antibody of 150,000 kD equals about 0.02 nMol) in 10 μ l serum under this concentration.In rabbit reticulocyte lysate is analyzed, can obtain to suppress fully with 6 μ g antibody/10 μ l human serums in the reaction.Use the inhibition of 1379 clones' Fab fragment check alternative pathway then.When 1379 clones' that use molar excess Fab, by this analysis and observation to the active inhibition fully of alternative pathway.

In the zymosan analysis, checked 1379 inhibition to come from the active ability of alternative pathway of the serum of a plurality of different mammalian species then.In the most of species that detected, 1379 antibody capables suppress alternative pathway activity (table 1) fully.Antibody capable suppresses the alternative pathway activity of the serum of mice, rat, people and several monkey classes fully.But, do not find any inhibition activity at Canis familiaris L. or Cavia porcellus.

Table 1

The species that alternative pathway is suppressed fully by mAb 1379
	● mouse ● people ● rat ● baboon ● macaque ● pig ● stump-tailed macaque ● horse
The species that alternative pathway is not suppressed by mAb 1379
	● Canis familiaris L. ● Cavia porcellus

The pharmacokinetics of 1379 antibody.Behind single injection inhibition type antibody, check mice with regard to the inhibition of alternative pathway at different time.1mg antibody causes and suppresses (Fig. 3) fully in IV injection 1 hour and IP injection 2 hours.The mice of accepting 1mg IP injection still kept the inhibition fully to alternative pathway in the time of 24 hours, the mice of accepting the 2mg injection still keeps fully when reaching 48 hours after injection and suppresses.The inventor also every other day injects 2mg 1379 antibody by peritoneal injection repeatedly, continues 14 days, and the result shows that 48 hours (not providing data) kept in the inhibition fully of injection back complement bypass at least the last time.These data clearly show that this mice mAb is not identified as " external " and supports its life-time service in vivo.At last, use the segmental experiment of antibody F (ab) to show that the inhibition of alternative pathway is the same with complete 1379 antibody and obtain almost to wait mol level (not providing data).

1379 epi-positions in conjunction with the BaSCR3 district.The ability of a series of B factor mutants of 1379 antibodies is existing to be described (Hourcade, 1995, J.Biol.Chem.), to characterize the mAb binding site.Experiment shows SCR2 and the SCR3 that some alanine displacement is introduced the people B factor, rather than SCR1, makes that 1379 antibody can not be in conjunction with the B factor.In these 25 kinds of different mutants that detected, 1379 basically in conjunction with B17 and B23 mutant, and keeps the binding ability be less than 20% itself and B18 mutant.These all are the SCR3 sudden changes.More specifically, mutant B17 replaces 139-Tyr-140-Cys-141-Ser with His-Cys-Pro, and this position is relevant with the one-tenth acquaintance B factor that is expressed as SEQ ID NO:2; Mutant B23 replaces 182-Glu-183-Gly-184-Gly-185-Ser with Gly-Asn-Gly-Val, and this position is also relevant with the one-tenth acquaintance B factor that is expressed as SEQ ID NO:2.Therefore, 1379 antibodies are to the Three S's CR domain of the B factor.

Conclusion

The inventor has prepared the novel monoclonal antibody at the mice B factor, is called as 1379 clones.This antibody is the specific inhibitor of complement bypass, and it suppresses this approach in vitro and in vivo fully.Single intraperitoneal injection 1 mg causes that the inhibition fully of alternative pathway reaches 48 hours, and multiple injection has prolonged the inhibition to this approach.

Use analyzed in vitro to show that 1379 have suppressed the alternative pathway activity of the serum of multiple species fully, these species comprise mice, rat, monkey and people.Use the binding affinity analysis of antagonist and a series of B factor mutants, record antigen site at the SCR3 of B factor domain (fB-/-the regional part of mice disappearance).This zone of B factor protein and D factor shearing site adjoin.1379 Fab fragment suppresses the active ability of alternative pathway and shows that it is not only to cause can prevent to be sheared sterically hindered by 1379, and this specific binding site still is the key position that the albumen of D factor mediation is sheared.1379 effectiveness at the serum of numerous different plant species like this illustrate that this site is conservative at the higher mammal camber.

Developed several other soluble complement inhibitor and it has been carried out identifying (Quigg; Weisman; Heller; Granger; Pratt), but believe that inhibitor described herein is first inhibitor that selectivity suppresses alternative pathway in animal species on a large scale.Suppress alternative pathway by selectivity, 1379 compare with the inhibitor that works in the C3 convertase level has several advantages.By not destroying classical pathway, this inhibitor may have less immunosuppressive effect.In addition, in fact the blocking-up of classical pathway can induce autoimmune.The selective exclusion of alternative pathway has improved the symptom (Watanabe) of lupus nephritis mouse model, and the C3 disappearance can not be improved its symptom.Alternative pathway is specifically related to multiple disease model (Thurman; Watanabe; Girardi), the treatment potential that has shown specificity alternative pathway inhibitor.

List of references:

1.Thurman et al.，2003，JImmunol170：1517-1523

2.Watanabe et al.，2000，JImmunol 164：786-794

3.Girardi et al.，2003，JClin Invest 112：1644-1654

4.Holers，V.M.2003，Clin Immunol 107：140-151

5.Densen et al.，l996，Mol Immunol 33：68(Abstract 270)

6.Matsumoto et al.，1997，Proc Natl Acad Sci USA94：8720-8725

7.Figueroa and Densen，1991，Clin Microbiol Rev 4：359-395

8.Quigg et al.，1998，J Immunol160：4553-4560

9.Weisman et al.，1990，Science 249：146-151

10.Heller et al.，1999，J Immunol163：985-994

11.Granger et al.，2003，Circulation 108：1184-1190

12.Pratt et al.，2003，Am JPathol 163：1457-1465

Embodiment 2

Following embodiment shows by the complement activation of alternative pathway very crucial in the progress of airway hyperreactivity and inflammation, shows further that also the inhibition to the complement activation of alternative pathway has suppressed airway hyperreactivity.

Consider the validity that suppresses complement activation before being exposed to anaphylactogen, the inventor has also detected the complement activation approach. Inventor's report is very crucial in the progress of airway hyperreactivity and airway inflammation by the activation of the complement cascade reaction of alternative pathway in this research.

Method

Animal

(Bar Harbor, ME) obtains the female C57BL/6 mouse in 8 to 12 ages in week from the Jackson laboratory. As previously mentioned, at first with the mouse of Factor B heterozygous deletion (fB+/-) and C57BL/6 incross, and then hand over mutually in F1 generation, hand over mutually then form fB-/-strain. In 7 generations then, backcrossed these mouse and C57BL/6 mouse. Use co-isogenic fB+ /+brood mouse mouse in contrast. C4 disappearance mouse ((C4 water), with C57BL/6 mouse 17 generations that backcrossed) is retained in Animal Lab.. The diet of employed all experiments were animal does not contain ovalbumin (OVA) in this research, and follows the management of laboratory animal in the medical treatment of national Jew and research center and the agreement of the use committee (Institutional Animal Care and Use Committee of the national jewish medical and research center) approval.

Experimental program

Be suspended in 2.25mg aluminium hydroxide (Alum Imuject the 1st day and 14 days by intraperitoneal injection (i.p.) 20 μ g; Pierce, Rockford, IL) OVA (V level, Sigma Chemical Co., St.Louis, MO) or artemisiifolia (Ambrosia artemisiifolia, Greer Laboratories, Lenoir, NC) the sensitization mouse, used then spraying OVA or artemisiifolia (1% at the 27th, 28 and 29 day, be dissolved in PBS), ultrasonic nebulizer (De Vibiss Health Care, Somerset, PA) attack 20 minutes every days by air flue.

Be reconstruct fB, each air flue attack not sensitization and sensitization fB-/-front 1 hour of mouse gives 10 μ g, 1 μ g or 0.1 μ g purifying fB (50 μ L are dissolved in PBS) by being used for nose. In contrast, give PBS.

In different experiments, attacked front 2 hours at each OVA, will give the sensitization mouse by i.p. injection (2mg/ processing/mouse) or by spray-on process for the antibody (anti-fB) of fB. With regard to spray-on process, 4 mouse are put into the lucite box, and use ultrasonic nebulizer (De Vilbiss Health Care) that the anti-fB of 0.5mg (being dissolved in 5ml PBS) is sprayed. In contrast, the rat IgG of same dose and volume injects or spraying by i.p. at same time point. At the 31st day, assessment AHR and on the same day with sacrifice of animal, collect BAL liquid, blood and lung tissue.

The purifying of Factor B

For reconstruct B-/-the alternative pathway activity of mouse, by affinity purification from normal mouse serum purifying mouse complement Factor B. According to the specification of manufacturer, by the anti-people's BF of goat (Diasorin, Stillwater, MN) being attached to CNBr-Activated Separose (Amersham, Arlington Heights, IL) preparation affinity column. By cardiac puncture the C57/B6J mouse is taken a blood sample, and collect blood in the syringe that contains 50 μ l 500mM EDTA, activate to prevent alternative pathway. With blood at 2000rpm centrifugal 15 minutes, collect then blood plasma. Then blood plasma is diluted with buffer solution (PBS, the pH7.4 that contain EACA 50 mM, EDTA 10 mM, benzamidine 2 mM) at 1: 1, and filter with 0.22 μ m filter (GE Water Technologies). Blood plasma is added affinity column, and wash with the buffer solution of 10 times of column volumes. With 5 M LiCl₂The wash-out Factor B is also used the PBS dialysed overnight. Electrophoresis is also with the purity of examining Ma Shi light blue staining examine Factor B on the 10%Tris-Glycine gel. Measure the concentration that (BioWhittaker, Inc., Walkersville, MD) measures LPS by TAL, find that this concentration is lower than 1 EU/mg purifying Factor B.

The formation of anti-Factor B antibody

Anti-mouse Factor B monoclonal antibody can be such as production as described in the embodiment 1. In brief, with the mouse of recombination fusion protein immunity Factor B disappearance, this recombination fusion protein is that the second and the 3rd short consensus repeat (SCR) domain and the immunoglobulin (Ig) by the Factor B gene forms. Select the SCR domain to be since they for fB-/-part of mouse Factor B gene delection section. Use then this protein immunization fB-/-mouse, and booster immunization four times, every three weeks of minor tick. Injection is merged splenocyte one day after with the myeloma cell who obtains from the much monoclonal antibodies of U.S.'s crolla center the last time. Identify then and characterize the clone of the anti-Factor B monoclonal antibody of secretion (mAb). A kind of hybridoma A1379 is used to these experiments. With Protein-G Sepharose post (Pharacia, Uppsala, Sweden) purifying A1379 from organize culture supernatant. Use polymyxins (Sigma-Aldrich, St.Louis, MO) that LPS is removed from the mAb of purifying. According to the specification of manufacturer, use TAL to measure (BioWhittaker, Inc., Walkersville, MD) and determine whether the LPS level of mAb is lower than 1 EU/mg mAb. Electrophoresis is also used the purity of examining Ma Shi light blue staining examine mAb on the 10%Tris-Glycine gel then.

The detection of air flue function

The variation of air flue function was assessed after airway reactivity increases (6.25,12.5,25,50 and 100mg/ml) gradually by giving concentration aerosolization acetonyl choline (MCh) 10 seconds (per minute is breathed 60 times, and tidal volume is 500 μ l) was attacked. (per minute breathing 160 times, tidal volume is that 150 μ l, end-expiratory positive pressure are 2-4cm H by mechanical ventilation for anesthetized mice (yellow Jackets, i.p., 70 to 90mg/kg), tracheotomy mouse (18G intubate)₂O) and assessed PFT (Takeda, 1997). By air-flow, volume and pressure substitution equation of motion are calculated (Labview, NationalInstruments, TX) airway resistance (RL) continuously. Get the maximum of RL, and be expressed as the change percentage in arid of the relative baseline of PBS aerosol acquisition. Baseline RL value does not have significant difference between separately disappearance mouse or control mice.

The measurement of bronchoalveolar lavage and cell factor

After having assessed the air flue function, by tracheal catheter Hank balanced salt solution (1 * 1ml, 37 ℃) lavation lung. Use cell counter (Coulter Counter; Coulter Co., Hialeah, FL) acquisition BAL cell number. The cell centrifugation preparation carried out the difference cell count and calculate percentage and the absolute number of every kind of cell type. By the cytokine levels (Tompkinson) in the ELISA assessment BAL liquid. Carry out IFN-γ, IL-4, IL-5, IL-10, IL-12 (all available from PharMingen, San Diego, CA) and IL-13 (R﹠D Systems, Minneapolis, MN) ELISA according to the indication of manufacturer.

Indication (Cedarlane Laboratories according to manufacturer, Hornby, Ontario, Canada), attacked rear 24 hours and for the third time and last the attack rear 24 hours and 48 hours at anaphylactogen for the first time or for the second time, measure the level of the C3a desArg in not the sensitization mouse and sensitization mouse BAL liquid by ELISA.

Histology and immunohistochemistry research

After obtaining BAL liquid, make atelectasis with 2ml 10% formalin by tracheae, fix by infusion process with same solution then. Histotomy dyes with h and E, periodic acid west husband's agent (PAS), and with the anti-mouse MBP of rabbit antibody (by J.J.Lee, Mayo Clinic, Scottsdale, AZ provides) cell that contains acidophilia major basic protein (MBP) is carried out the immunohistochemistry detection. By the blindness mode checked section and by use NIHScion Image software (1.62 editions by U.S.'s National Institutes of Health exploitation, can get on the internet) respectively eosinophil and the calyciform cell of peribronchial tissue are analyzed.

The measurement of total IhE and OVA specific antibody

Such as (Tompkinson) as described in the forefathers, measured the serum levels of total IgE and the special IgE of OVA and IgG1 by ELISA. The OVA specific antibody titre of sample is relevant with the standard items of internal gathering, they by the people for being appointed as 500 ELISA units (EU). By with known mouse IgE standard items (553481, PharMingen) relatively calculate the level of total IgE.

Statistical analysis

Variance analysis (ANOVA) is used to detect the level of difference between all groups time. Undertaken whole groups of inferior comparisons by the reliable significant difference of Tukey-Kramer (HSD). The probable value of conspicuousness is set as 0.05. All measured value is represented as the standard error (SEM) of mean value ± mean value.

The result

In the progress of complement activation by alternative pathway AHR after anaphylactogen is attacked the sensitization mouse Very important

Be the effect of assessment alternative pathway in the progression of AHR and airway inflammation, the OVA sensitization and not sensitization fB-/-mouse and matched control mouse (fB+ /+) be with the continuously attack three days of 1%OVA aerosol. By sensitization with attack fB+ /+mouse and the fB+ that is only attacked /+mouse is compared its reactivity to MCh increases (Fig. 4). On the contrary, by sensitization with attack fB-/-mouse with by sensitization and attack fB+ /+mouse compares it and shows in whole dose-response linearity curve the significantly reduced reactivity of MCh (P＜0.01), therefore show after by sensitization and attack obviously AHR can not occur.

Very important in the progress of the airway inflammation after anaphylactogen is attacked the sensitization mouse of the complement activation by alternative pathway

Airway inflammation is the characteristic features of allergic airway diseases. Be the assessment airway inflammation, air flue is attacked and was obtained BAL liquid and lung tissue in rear 48 hours the last time. By sensitization with attack fB+ /+total cell count that mouse is compared with the mouse of only being attacked in its BAL liquid increases, particularly the quantity of eosinophil increases, the mouse of wherein only being attacked does not have eosinophil (Fig. 5) in its BAL liquid. By sensitization with attack fB-/-mouse with by sensitization and attack fB+ /+total cell count and eosinophil quantity that mouse is compared in the BAL liquid significantly reduces (p＜0.01), still still is significantly higher than the control group (p＜0.01) of only being attacked. Equally, fB-/-mouse compares with the control group of artemisiifolia sensitization and attack in the eosinophil quantity in the BAL liquid with the artemisiifolia sensitization and after attacking and also reducing (Fig. 5).

The anaphylactogen sensitization is attacked with air flue and is only compared the increase that can cause peribronchitis disease by attack, particularly the increase (Fig. 3) of eosinophil infiltration. But by sensitization with attack fB-/-mouse with compared peribronchitis disease by sensitization and attack control mice and significantly reduce (table 2). Be the eosinophil infiltration capacity in the quantitative lung, histotomy is not with anti-major basic protein dye (providing data). In the mouse of only being attacked, in peribronchial tissue, only find a small amount of eosinophil. The fB+ that sensitization and anaphylactogen are subsequently attacked /+mouse so that its bronchus week eosinophil quantity significantly increase (table 2). On the contrary, by sensitization and attack fB-/-only have a small amount of eosinophil to infiltrate (table 2) in its bronchus week of mouse.

Another characteristics of allergic airway diseases are human airway epithelial cells calyciform cell hyperplasia. Lung is with periodic acid-Xi Fu agent dyeing, to identify the cell that contains mucus in the airway epithelia. A large amount of cells by sensitization and attack mouse are found to be the mucus positive (table 2) by dyeing, on the contrary, do not detect PAS positive cell (table 2) in the mouse of only being attacked. By sensitization and attack fB-/-cell that contains mucus in its airway epithelia of mouse significantly is lower than by sensitization and attacks wild-type mice (p＜0.001).

Affect the generation of the cell factor in the BAL liquid by the complement activation of alternative pathway

The Th2 cell factor that the T cell produces plays an important role in induced hypersensitivity airway inflammation and AHR. Attack rear cytokine response for the assessment anaphylactogen, OVA attacks the concentration of assessing IL-4, IL-5, IL-10, IL-12, IL-13 and IFN-γ in the BAL liquid in rear 48 hours the last time. Compare with the control group of only being attacked, significantly improved (p＜0.05) by its IL-4 of the wild-type mice of sensitization and attack, IL-5 and IL-13, and IL-10, IL-12 and IFN-γ significantly reduce (p＜0.05) (not providing data). FB-/-T in the BAL liquid of mouse _H2 cytokine levels (IL-4, IL-5 and IL-13) decrease (not providing data).

The fB disappearance does not affect the serum levels of antigen-specific antibody

Last air flue is attacked the serum levels of measuring total IgE and the special IgE of OVA and IgG1 in rear 48 hours. Compare with the control group mice of only being attacked, by the fB+ of sensitization and attack /+level of its total IgE of mouse and the special IgE of OVA and IgG1 increase (table 3). Equally, fB-/-level of its total IgE of mouse and the special IgE of OVA and IgG1 also increases, this with by the fB+ of sensitization and attack /+mouse do not have statistically difference, shows in these mouse being remained intact by the humoral response of anaphylactogen sensitization and attack.

Table 2. calyciform cell hyperplasia and bronchus week Eosinophilic Inflammation quantitatively

	F β+/+only attacked	F β+/+by sensitization and attack	F β-/-by sensitization and attack	C4+ /+by sensitization and attack	C4-/-by sensitization and attack	Contrast A b is by sensitization and attack	Anti-f β, i.p. is by sensitization and attack	Anti-f β ncb is by sensitization and attack
									PAS positive cell (cell/mm BM) α MBP positive cell (cell/mm BM)	N.D.     1.3±0.9	132±35 *     80±16 *	36±19#     24±14#	156±27 *     95±20 *	149±15 *     87±21 *	153±33 *     95±28 *	28±10#     20±9#	43±12#     33±16#

As described in the method part, sensitization and attack mouse. Attack the last time the quantity of estimating calyciform cell (PAS positive cell) and bronchus week eosinophil (anti-major basic protein (MBP) positive cell) in rear 48 hours. Be expressed as mean value ± SEM; F β-/-: Factor B lacks mouse; F β+/+: congeric strains wild type control mice; C4-/-: complement factor 4 lacks mouse; C4+ /+: congeric strains wild type control mice; Contrast Ab: processed by i.p. with contrast Ab by the C57BL/6 mouse of sensitization and attack; Anti--f β, i.p.: by the C57BL/6 mouse of sensitization and attack by i.p with anti-f β antibody treatment; Anti-f β neb: passed through to suck with anti-f β antibody treatment by the C57BL/6 mouse of sensitization and attack; BM (basement membrane).^*P＜0.05, with the f β that is only attacked+/+, by the f β that attacked-/-, compare by the anti-f β i.p. of sensitization and attack and by the anti-f β neb of sensitization and attack. #p＜0.05: with the f β that is only attacked+/+compare.

Table 3: serum immunoglobulin level

	F β+/+only attacked	F β+/+by sensitization and attack	F β-/-only attacked	F β-/-by sensitization and attack	C57BL/6 is by sensitization and attack	Ab is by sensitization and attack in contrast	Anti-f β, i.p. is by sensitization and attack	Anti-f β ncb is by sensitization and attack
									Total special IgG1 of the special IgE of IgE (ng/ml) OVA (EU/ml) OVA (EU/ml)	47.3±11.2     ＜10     ＜10	219.8±48.4 *   145.1±36.7 *   189.1±20.5 *	38.5±12.5     ＜10     ＜10	198.3±31.2 *   166.2±42.1 *   155.9±38.8 *	241.1±37.6 *   153.8±47.4 *   171.8±71.1 *	238.5±41.1 *   128.1±30.8 *   146.5±61.2 *	189.6±33.2 *   99.1±27.4 *   106.5±28.9 *	229.5±44 *   122.5±39.5 *   120.1±30.8 *

As described in the method part, sensitization and attack mouse. Attack the last time the rear serum levels of estimating immunoglobulin (Ig) in 48 hours. Be expressed as mean value ± SEM; F β-/-: Factor B lacks mouse; F β+/+: congeric strains wild type control mice; Contrast Ab: processed by i.p. with contrast Ab by the C57BL/6 mouse of sensitization and attack; Anti--f β, i.p.: by the C57BL/6 mouse of sensitization and attack by i.p. with anti-f β antibody treatment; Anti-f β neb: passed through to suck with anti-f β antibody treatment by the C57BL/6 mouse of sensitization and attack; EU/ml (Elisa unit/ml).^*P＜0.05, with the f β that is only attacked+/+and by the f β that attacked-/-compare.

The activation of classical pathway is unimportant to the progress of allergic airway diseases in this model

For further determining by the very crucial complement pathway of anaphylactoid progress in the lung of the mouse of sensitization and attack, the inventor uses disappearance complement component 4 (C4-/-, it is necessary) in the activation of classical pathway and lectin pathway mouse and fB-/-mouse compares.

For the activation of assessment complement pathway, estimated the C3a desArg level in the BAL liquid. The mouse C3a desArg level lower (not providing data) of only being attacked. On the contrary, for the first time, for the second time and for the third time attack after, increased to some extent by C3a desArg level in the sensitization mouse BAL liquid, and attack the last time and reached peak (not providing data) in rear 48 hours. What is interesting is, by sensitization and attack C4-/-mouse be on close level by sensitization and the C3a that attacks wild-type mice, this with by sensitization and attack fB-/-mouse is opposite: fB-/-mouse compares C3a desArg level reduction (not providing data) with its wild-type mice by sensitization and attack separately. These data show after the sensitization mouse is exposed to anaphylactogen, the complement activation by alternative pathway occurred.

C4-/-mouse (occur with by sensitization and attack C4+ /+the suitable AHR level (Fig. 9) of mouse. Equally, with by sensitization with attack control mice (n=10; Be respectively 175 ± 53; 35 ± 12; 115 ± 32 * 10³Cell) compare, C4-/-total cell count (mean value ± SEM, n=10 in mouse bronchial alveolar wass (BAL) liquid; 163 ± 35 * 10³Cell) or lymphocyte quantity (28 ± 9 * 10³Cell) and eosinophil (98 ± 23 * 10³Cell) quantity does not reduce (not providing data). With separately by sensitization with attack the WT mouse and compare, further by sensitization and attack C4-/-its of mouse in bronchus week eosinophil quantity and calyciform cell quantity improve the standard quite (table 2). These discoveries show that the activation of the classical pathway in this model is unimportant for the progress of allergic airway diseases.

In fB disappearance mouse AHR does not appear and airway inflammation is not that OVA is special

For be evaluated at the anaphylactogen sensitization and attack after whether airway hyperreactivity do not occur be because OVA is not had special replying, with the artemisiifolia sensitization and attack fB-/-mouse and wild-type mice. The fB-of artemisiifolia sensitization and attack/-mouse reduces the reactivity of MCh, and fB+ /+to MCh strong reaction (Fig. 6 A and 6B). Equally, with fB+ /+compare, the fB-of artemisiifolia sensitization and attack/-mouse BAL liquid in airway inflammation reduce (Fig. 6 C).

Give Factor B reconstruct fB-/-ability of AHR and airway inflammation appears in mouse

Be reconstruct Factor B in lung, before each air flue is attacked, fB-/-accept the single nose to use 10 μ g, 1 μ g or 0.1 μ g purifying Factor B (Fig. 7) or PBS. Before each the attack with 0.1 μ gB factor treatment by sensitization and attack fB-/-mouse its to the decline of MCh reactivity, this to process with PBS by sensitization and attack fB-/-mouse is similar, but with by sensitization and attack fB+ /+mouse compares remarkable reduction (Fig. 7 A). With process with PBS or 0.1 μ g purifying Factor B by sensitization and attack fB-/-mouse compares, and with being strengthened to some extent by sensitization and its airway reactivity of attack mouse that 1 μ g purifying Factor B is processed, still do not have statistically difference (Fig. 7 A). On the contrary, with by sensitization and attack fB+ /+mouse is identical, before each air flue is attacked with 10 μ g purifying Factor Bs process by sensitization and attack fB-/-its reactivity enhancing to MCh of mouse.

With by sensitization and attack fB+ /+mouse in viewed cell number identical, before each air flue is attacked with 10 μ g purifying Factor Bs process by sensitization and attack fB-/-mouse, also improved airway inflammation, particularly improve the eosinophil number in the BAL liquid, but processed the quantity (Fig. 7 B) that can not improve the eosinophil in the BAL liquid with 0.1 μ g or 1 μ g purifying Factor B. If before each air flue is attacked, give not sensitization acceptor with Factor B, do not observe AHR or Airway inflammatory response, this shows needs the sensitization stage so that attack is made a response, be further illustrated in simultaneously fB-/-mouse in the sensitization stage be complete. FB-/-these data of mouse show that directly the Factor B of alternative pathway is crucial for the progress of allergic airway diseases.

Process the appearance that suppresses by the AHR of sensitization and attack mouse with the fB neutralizing antibody

For being evaluated at the effect of being passed through the complement activation of alternative pathway in the mouse of the no gene delection of sensitization and attack, described with the C57BL/6 sensitization according to the method part. 1379 anti-Factor B antibody described in the embodiment 1 are given by spray-on process whole body or part, and this has been shown that it is the effective way that gives other complement inhibitor. Normal mouse, after phase of the attack was processed by whole body or part (spray-on process) with anti-Factor B after by sensitization, its AHR significantly reduced (Fig. 8 A and 8B), simultaneously airway inflammation and the eosinophilia suppressed (Fig. 8 C) in the air flue. In addition, tissue inflammation, all eosinophil quantity (table 2) of bronchus and calyciform cell quantity (table 2) reduce in the mouse that these anti-fB process. In addition, IL-4, IL-5 and IL-13 level significantly reduce (not providing data) in the BAL liquid of the mouse of fB antibody treatment. Equally, with anti-Factor B process by sensitization and attack C4-/-mouse reduced its airway reactivity and airway inflammation (Figure 10). These results are consistent with the research of using complement inhibitor: as broad as long between classical pathway and alternative pathway, but blocked airway reactivity and the progress of AHR in late period.

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Embodiment 3:

The following examples a series of anti-Factor B antibody that the inventor is produced described other in conjunction with data. Analyze for combination and/or the inhibition of checking mouse Factor B and Human complement factor B to various antibody. As can be seen, mAB 1379 can in conjunction with and suppress mouse and people's Factor B. On the contrary, being called 624 mAb can be in conjunction with mouse and Human complement factor B, but can not suppress people's alternative pathway. Competitive ELISA is used for further assessment antibody. As can be seen, antibody 624,691 and 1231 can not be blocked 1379 combination. Therefore these antibody one fix on different sites in conjunction with this albumen, and this has explained why they are in external its function that do not suppress in conjunction with Factor B. But antibody 395,1322 and 1060 is competitive inhibitors of 1379.

Table 4

The clone	Isotype	In conjunction with mouse fB	In conjunction with people fB	Suppress mouse alternative pathway (zymosan analysis)	Suppress people's alternative pathway (rabbit reticulocyte lysate analysis)	With 1379 competitive binding people fB
							1379	IgG1 K	+++	+++	+++	+++	+++
395	IgG1 K	+++	++	++	+++	+++
							1322	IgG2b K	+++	+++	+	++	+++
624	IgG1 K	+++	+++	+	-	-
							691	IgG1 K	+++	+++	+	-	-
1060	IgG2b K	+++	+++	+	++	++
							1231	IgG1	+++	+++	+	-	-
E1128		-	+++	-	0	NA

Embodiment 4

Following embodiment has described the epi-position figure of mAb 1379 on the Human complement factor B surface.

First experiment to mAb 1379 antibody tabulation bitmaps shows that epi-position or antibody combining site on the Factor B are not linear. When antibody was attached on the elisa plate, it was combined with full-length proteins strongly. Having made up length is that 10 amino acid whose peptides are to cross over the albumen zone (SCR2-3) of known appearance combination. When these peptides are attached on the elisa plate, antibody and nonrecognition they, show that neither one is identified as epi-position in these linear orders.

The conservative mating surface of the expectation of the Human complement factor B that will be identified by mAb 1379 or epi-position modelling. In brief, the tertiary structure (Protein Data Bank (PDB) id 1GHQ) take the three-dimensional structure of the CR2-SCR1-2 that resolves as the fundamental construction Human complement factor B. Final mask is improved to the minimum energy state, and constraint is fixed on four absolute conservative Cys residues of each SCR. The homogeneity of the sequence of Factor B SCR2-3 and CR2 SCR1-2 is 30% (the highest), with H factor S CR15-16 be 25%, with CD46 be 20%. Figure 11 illustrates the model of Factor B structure, and has pointed out the amino acid position of corresponding mAb 1379 epi-positions (with respect to SEQ ID NO:2). The residue that is considered to form the comformational epitope of mAb 1379 antibody is Ala137, Tyr139, Ser141, Glu182, Ser185, Thr189, Glu190 and Ser192, but this epi-position may only contain a small amount of residue shown in Figure 11, basically all residues or the more residues that go out more shown in this Fig.

Now this model discuss before being used to predict from Hourcade (Hourcade, 1995, J. The effect of Factor B mutant Biol.Chem), this mutant are used for characterizing the epi-position (referring to embodiment 1) in conjunction with mAb 1379 antibody at first. Hereinafter illustrate from these a series of four kinds of mutant, they contain the residue of the mAb 1379 epi-position models that belong to shown in Figure 11. Shown in hereinafter, the B17 of the combination of the reduction mAb 1379 shown in the embodiment 1 and B23 mutant have the particular permutation (being represented as overstriking and inclination) of estimating towards inside, therefore may disturb in conjunction with the epi-position of mAb 1379 or the structure of conservative mating surface. Although mutant B16/17 contains in conjunction with the residue in the modelling epi-position of mAb 1379, it does not think the sudden change with interference table bit architecture, this may be interpreted as what in first collection of illustrative plates experiment this mutant in conjunction with this antibody. Equally, although mutant B23/24 also contains in conjunction with the residue in the modelling epi-position of mAb 1379, this mutant also is combined in the antibody in the first collection of illustrative plates experiment, and the residue that forms antibody contact site may not can be disturbed by these sudden changes. Antibody of the present invention has also been illustrated in this experiment can in conjunction with Factor B albumen or its part, have the conservative sudden change that suddenlys change or substantially do not disturb this epi-position.

B17：Y139-C140-S141

Displacement: H P

B23：E182-G183-G184-S185

Displacement: G N V

Bl6/17：G136-A137-G138

Displacement: N S S

B23/24：S187-G188-T189-E190-P191-S192

Displacement: D E T A V

Figure 12 is schematic diagram, and this illustrates the modelling compound of the mAb1379 (a Fab fragment) in conjunction with Factor B, and the antigen binding end of this Fab is modeled to cover whole epitope regions (as shown in figure 11) of drawing.

Embodiment 5

Following embodiment shows that the inhibition of the inhibition of complement bypass, particularly Factor B suppresses and the prevention animal avoids renal ischemia-reperfusion injury.

Test to check the validity of mAb1379 aspect the reduction damage in the ischemic Acute Renal Failure model. In this model, by with mouse anesthesia and with renal pedicle clam 24 minutes, induce ischemic Acute Renal Failure. Injection 1mg mAb induced damage after one hour in the mouse peritoneum. The method causes the reverse form of ischemic Acute Renal Failure, and the damage peak generally occurred after clip is taken from the kidney base of a fruit and blood backflow to kidney in 24 hours. Then by measuring such as the accumulation of the nitrogenouz wastes of SUN and serum creatinine and the morphology lesion assessment kidney injury of being assessed kidney by the Pathological scholar. The inventor is further illustrated in the alternative pathway that kidney pours into rear complement again and is activated very soon, shows that also this activation helps caused kidney injury.

Immunofluorescence technique and western blot analysis detect confirms that 1379 antibody described herein have effectively stoped complement activation behind I/R. As shown in figure 13, compare its plasma urea nitrogen (SUN) increase rate littler (78 ± 15mg/dL to 119 ± 15mg/dL, p＜0.05, every group of n=11) after pouring into again 24 hours with the wild type control group with 1379 pretreated mouse. In with 1379 mouse of processing, slighter Histological injury is arranged also. During by virologist's classification, 1379 process mouse significantly reduces (3.3 ± 0.5 pairs 4.9 ± 0.1, p＜0.01, every group of n=10) than its tubular injury of control mice with the blindness form. Therefore, 1379 have effectively prevented the complement activation in the mouse kidney behind I/R, and function damage and Histological injury behind the I/R have been alleviated in 1379 preliminary treatment.

In another experiment, by cultivating the renal cells chemical hypoxia that makes in the culture with antimycin two hours. Then cell is exposed to fresh mice serum (as the complement source), measures the lactic dehydrogenase (LDH) as cell death marker, and use business analysis kit (Promega, Madison, WI) in arbitrary unit, to express. Be exposed to LDH that cell in antimycin and the serum more only is exposed to its release of cell in the serum and increase significantly that (100,140 ± 3307 (antimycin+serum) are to 69,255 ± 9754, p＜0.05; Do not provide data). But when serum before cell is cultivated was hatched with mAb1379, the release of LDH dropped to 76,471 ± 7720 (p＜0.01 pair cell with antimycin+serum processing does not provide data). Therefore mAb1379 protected in vivo or the external component that is exposed to alternative pathway in contain the low renal cells of oxygen.

Each piece list of references that this paper quotes is included this paper in full by quoting. By quoting each piece U.S. Provisional Application NO.60/543,594, U.S. Provisional Application NO.60/636,239 and whole this paper that openly include in of PCT application NO.PCT/US2004/015040.

Although each embodiment of the present invention is described in detail, it is evident that: those skilled in the art can make amendment and changes these embodiments. But should know to be appreciated that: these modifications and change are in the scope of the present invention that claims limit.

Sequence table

＜110〉V.M. Horace

J.M. plucked instrument is graceful

C. the cloth of making pottery

E.W. Ge Erfande

G.S. Gilderson

＜120〉inhibition of the B factor, complement bypass and relevant therewith method

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325 330 335

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

340 345 350

Lys Arg Ala Leu Gln Ala Val Tyr Ser Met Met Ser Trp Ala Gly Asp

355 360 365

Ala Pro Pro Glu Gly Trp Asn Arg Thr Arg His Val Ile Ile Ile Met

370 375 380

Thr Asp Gly Leu His Asn Met Gly Gly Asn Pro Val Thr Val Ile Gln

385 390 395 400

Asp Ile Arg Ala Leu Leu Asp Ile Gly Arg Asp Pro Lys Asn Pro Arg

405 410 415

Glu Asp Tyr Leu Asp Val Tyr Val Phe Gly Val Gly Pro Leu Val Asp

420 425 430

Ser Val Asn Ile Asn Ala Leu Ala Ser Lys Lys Asp Asn Glu His His

435 440 445

Val Phe Lys Val Lys Asp Met Glu Asp Leu Glu Asn Val Phe Tyr Gln

450 455 460

Met Ile Asp Glu Thr Lys Ser Leu Ser Leu Cys Gly Met Val Trp Glu

465 470 475 480

His Lys Lys Gly Asn Asp Tyr His Lys Gln Pro Trp Gln Ala Lys Ile

485 490 495

Ser Val Thr Arg Pro Leu Lys Gly His Glu Thr Cys Met Gly Ala Val

500 505 510

Val Ser Glu Tyr Phe Val Leu Thr Ala Ala His Cys Phe Met Val Asp

515 520 525

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

530 535 540

Leu Glu Ile Glu Glu Val Leu Phe His Pro Lys Tyr Asn Ile Asn Gly

545 550 555 560

Lys Lys Ala Glu Gly Ile Pro Glu Phe Tyr Asp Tyr Asp Val Ala Leu

565 570 575

Val Lys Leu Lys Asn Lys Leu Lys Tyr Gly Gln Thr Leu Arg Pro Ile

580 585 590

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

595 600 605

Thr Ala Thr Cys Lys Gln His Lys Glu Gln Leu Leu Pro Val Lys Asp

610 615 620

Val Lys Ala Leu Phe Val Ser Glu Gln Gly Lys Ser Leu Thr Arg Lys

625 630 635 640

Glu Val Tyr Ile Lys Asn Gly Asp Lys Lys Ala Ser Cys Glu Arg Asp

645 650 655

Ala Thr Lys Ala Gln Gly Tyr Glu Lys Val Lys Asp Ala Ser Glu Val

660 665 670

Val Thr Pro Arg Phe Leu Cys Thr Gly Gly Val Asp Pro Tyr Ala Asp

675 680 685

Pro Asn Thr Cys Lys Gly Asp Ser Gly Gly Pro Leu Ile Val His Lys

690 695 700

Arg Ser Arg Phe Ile Gln Val Gly Val Ile Ser Trp Gly Val Val Asp

705 710 715 720

Val Cys Arg Asp Gln Arg Arg Gln Gln Leu Val Pro Ser Tyr Ala Arg

725 730 735

Asp Phe His Ile Asn Leu Phe Gln Val Leu Pro Trp Leu Lys Asp Lys

740 745 750

Leu Lys Asp Glu Asp Leu Gly Phe Leu

755 760

Claims (46)

1. a separation antibody or its Fab, the 3rd short consensus repeat (SCR) domain of its selective binding B factor, wherein this antibody stops the formation of C3bBb complex.

2. the separation antibody of claim 1 or its Fab, wherein this antibody or its Fab are in conjunction with the B factor and prevention or suppress the D factor and shear the B factor.

3. the separation antibody of claim 1 or its Fab, wherein this antibody or Fab are in conjunction with the 3rd short consensus repeat (SCR) domain of the people B factor.

4. the separation antibody of claim 1 or its Fab, wherein this antibody or its Fab selective binding are selected from the epi-position in the Three S's CR domain of the following B factor:

(a) comprise the epi-position of the B factor of at least a portion people B factor (SEQ ID NO:2), comprise from about Tyr139 position to about Ser185 position, or its equivalent position in non-human B factor sequence;

(b) comprise the epi-position of the B factor of at least a portion people B factor (SEQ ID NO:2), comprise from about Try139 position to about Ser141 position, or its equivalent position in non-human B factor sequence;

(c) comprise the epi-position of the B factor of at least a portion people B factor (SEQ ID NO:2), comprise from about Glu182 position to about Ser185 position, or its equivalent position in non-human B factor sequence;

(d) comprise the epi-position of the B factor of at least a portion people B factor (SEQ ID NO:2), comprise any one or a plurality of column position or its equivalent position in non-human B factor sequence: Tyr139, Cys140, Ser141, Glu182, Gly184 or Ser185 down.

5. the separation antibody of claim 1 or its Fab, wherein the B factor of this antibody or the multiple mammalian species of its Fab selective binding.

6. the separation antibody of claim 1 or its Fab, wherein this antibody or its Fab selective binding people and be selected from the B factor of following animal: the primates except that the mankind, mice, rat, pig, horse and rabbit.

7. the separation antibody of claim 1 or its Fab, wherein this antibody is the isotype or the subclass of non-complement activation.

8. the separation antibody of claim 1 or its Fab, wherein this antibody is monoclonal antibody.

9. the separation antibody of claim 1 or its Fab, wherein this Fab is the Fab fragment.

10. the separation antibody of claim 1 or its Fab, wherein this antibody is humanized antibody.

11. the separation antibody of claim 1 or its Fab, wherein this antibody is bi-specific antibody.

12. the separation antibody of claim 1 or its Fab, wherein this antibody is univalent antibody.

13. the separation antibody of claim 1 or its Fab, wherein this antibody is monoclonal antibody 1379 (being produced by ATCC preserving number PTA-6230).

14. a separation antibody or its Fab, its selective binding are from the B factor of multiple mammalian species, wherein this antibody has stoped the formation of C3bBb complex.

15. the separation antibody of claim 14 or its Fab, wherein this antibody or its Fab selective binding people and the B factor that is selected from following animal: the primates except that the mankind, mice, rat, pig, horse and rabbit.

16. the separation antibody of claim 14 or its Fab, wherein this antibody is the isotype or the subclass of non-complement activation.

17. the separation antibody of claim 14 or its Fab, wherein this antibody is monoclonal antibody.

18. the separation antibody of claim 14 or its Fab, wherein this Fab is the Fab fragment.

19. a separation antibody or its Fab, its selective binding B factor, wherein this antibody or its fragment competitive inhibition monoclonal antibody 1379 (being produced by ATCC preserving number PTA-6230) combine with the specificity of the people B factor, and wherein, when this antibody or its Fab during in conjunction with the people B factor, the ability that monoclonal antibody 1379 suppresses complement bypasses is suppressed.

20. the antibody of claim 19 or its Fab, wherein combining of this antibody or its Fab competitive inhibition monoclonal antibody 1379 and the people B factor detected relatively binding specificity by antibody-antibody competition analysis when the people B factor exists.

21. a separation antibody or its fragment, its selective binding people B factor, wherein anti-or its Fab of this separation antibody or its fragment competitive inhibition two combines with the specificity of the people B factor, and wherein this two resist or its Fab in conjunction with the Three S's CR domain of the people B factor.

22. a compositions comprises among the claim 1-21 each separation antibody or its Fab.

23. an antigen-binding polypeptides, the 3rd short consensus repeat (SCR) domain of its selective binding B factor, wherein this antigen-binding polypeptides has stoped the formation of C3bBb complex.

24. an antigen-binding polypeptides, the B factor of the multiple mammalian species of its selective binding, wherein this antigen-binding polypeptides has stoped the formation of C3bBb complex.

25. a compositions comprises the antigen-binding polypeptides of claim 23 or claim 24.

26. one kind is reduced or the airway hyperreactivity (AHR) of prevention animal or the method for airway inflammation, comprises suffering from inflammation relevant airway hyperreactivity or airway inflammation or the animal that suffers from this disease risk being arranged according to each antibody or its Fab in the claim 1 to 18.

27. the method for claim 26, wherein this antibody or Fab give animal with certain amount, with give this antibody or Fab before compare, this amount can effectively significantly reduce the airway hyperreactivity of animal.

28. the method for claim 26, wherein this antibody or Fab give animal with certain amount, compare with suffering from the level of airway hyperreactivity that inflammation do not give the animal population of this antibody or Fab, this amount can effectively significantly reduce the airway hyperreactivity of animal.

29. the method for claim 26, wherein this antibody or Fab has reduced animal to the acetonyl choline or to the reactivity of histamine.

30. the method for claim 26, wherein this antibody or Fab give with being selected from following pharmaceutically suitable carrier: can disperse dry powder, dehydrated alcohol, Caplet, liposome, atomisation agent and injectable excipient.

31. the method for claim 26, wherein this antibody or Fab give in being selected from following carrier or device: dehydrated alcohol, dry powder intake system, ultrasonic intake system, pressure measurement inhaler and metering solution device.

32. the method for claim 26, wherein said antibody or Fab give described mammal with being selected from following reagent: the inhibitor of corticosteroid, beta-2-agonists (long-acting or fugitive), leukotriene instrumentality, antihistamine, phosphodiesterase inhibitor, sodium cromoglicate, nedocromil, theophylline, cytokine antagonist, cytokine receptor antagonist, anti-IgE and T cell function.

33. the method for claim 26, wherein said airway hyperreactivity or airway inflammation and be selected from following disease association: asthma, chronic obstructive disease of lung (COPD), anaphylaxis broncho-pulmonary aspergillosis, hypersensitivity pneumonitis, the eosinophilic granulocyte pneumonia, emphysema, bronchitis, allergic bronchitis, bronchiectasis, cystic fibrosis, tuberculosis, hypersensitivity pneumonitis, occupational asthma, sarcoidosis, RADS, interstitial lung disease, hypereosinophilic syndrome, rhinitis, sinusitis, exercise-induced asthma, asthma is brought out in pollution, cough variant asthma, parasitic disease of lung, respiratory syncytial virus (RSV) infects, parainfluenza virus (PIV) infects, rhinovirus (RV) infects and adenovirus infection.

34. the method for claim 26, wherein this airway hyperreactivity is relevant with allergic inflammation.

35. the method for claim 26, wherein this (AHR) or airway inflammation are relevant with asthma.

36. the method for claim 26, wherein this (AHR) or airway inflammation are relevant with COPD.

37. one kind is reduced or the airway hyperreactivity (AHR) of prevention animal or the method for airway inflammation, comprises that the reagent that selectivity is suppressed complement bypass suffers from inflammation relevant airway hyperreactivity or airway inflammation or the animal that suffers from this disease risk is arranged.

38. a method that reduces or prevent the ischemia reperfusion injury of animal comprises and will suffer from ischemia reperfusion injury or the animal that suffers from this disease risk is arranged according to each antibody or its Fab in the claim 1 to 18.

39. the method for claim 38, wherein this ischemia reperfusion injury is the kidney ischemia reperfusion injury.

40. the method for claim 38, wherein this antibody or Fab give animal with certain amount, compare with not giving this antibody or Fab, and this amount can effectively significantly suppress the rising of the serum urea nitrogen of animal.

41. the method for claim 38, wherein this antibody or Fab give animal with certain amount, and do not give antibody or Fab is compared, and this amount can effectively significantly reduce the Histological injury to the renal tissue of animal.

42. the method for claim 26 or 38, wherein this antibody or Fab give by being selected from following approach: in oral, nose, part, suction, the trachea, percutaneous, rectum and parenteral route.

43. one kind is reduced or the airway hyperreactivity (AHR) of prevention animal or the method for airway inflammation, comprises suffering from inflammation relevant airway hyperreactivity or airway inflammation according to the antigen-binding polypeptides of claim 23 or claim 24 or the animal that suffers from this disease risk being arranged.

44. a method that reduces or prevent the ischemia reperfusion injury of animal comprises and the antigen-binding polypeptides of claim 23 or claim 24 is suffered from ischemia reperfusion injury or the animal that suffers from this disease risk is arranged.

45. each method among the claim 26-44, wherein this animal is a mammal.

46. each method among the claim 26-44, wherein this animal is behaved.

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