CN104159600A - Targeting interferon-lambda with antibodies potently enhances anti-tumor and anti-viral activities - Google Patents

Abstract

The present invention concerns methods and compositions for forming complexes of interferon-lambda with an antibody or antigen-binding antibody fragment. In preferred embodiments, the interferon-lambda and the antibody or fragment are fusion proteins, each comprising a dimerization and docking domain (DDD) moiety from human protein kinase A or an anchor domain (AD) moiety from an A-kinase anchoring protein (AKAP). In more preferred embodiments, the interferon-antibody complex is more efficacious for treatment of cancer, asthma, Alzheimer's disease, multiple sclerosis or viral infection than interferon-lambda alone, antibody alone, or the combination of unconjugated interferon-lambda and antibody.

Description

With antibody target interferon-λ, effectively strengthen antitumor and antiviral activity

Related application

The application has required the interim U.S. Patent application 61/591,087 of submitting on January 26th, 2012 and the U.S. Patent application No.13/412 submitting on March 6th, 2012,816 rights and interests according to 35U.S.C.119 (e).The mode that the text of above-cited each priority requisition is quoted is in full incorporated herein.

Sequence table

The application comprises by the sequence table that EFS-Web submits to and the mode hereby quoted is in full incorporated to.Described ASCII copy creating was on January 22nd, 2013, and being named as IBC136WO.txt and size is 50,753 bytes.

Background

Invention field

The present invention relates to comprise and be connected to the interferon-λ (IFN-λ) of antibody or antigen binding antibody fragment, the more preferably DOCK-AND-LOCK of IFN-λ 1 ^tM(DNL ^tM) complex compositions and treatment using method.In preferred embodiments, antibody can be anti-TROP-2, anti-CEACAM5, anti-CEACAM6, anti-HLA-DR, anti-stick albumen, anti-CD19, anti-CD20, anti-CD74, anti-AFP or anti-CD22 antibody.Yet, those skilled in the art will recognize that the present invention is not subject to so restriction and more broadly contains antibody-interferon compound.Preferably, described DNL ^tMcomplex is to use as U.S. Patent No. 7,521,056,7,527,787,7,534,866,7,550,143 and 7,666, and shown in 400, the compositions of example and technology make, and described patent embodiment separately is partly incorporated herein by reference.The interferon that antibody yoke closes retains external activity and shows the serum half-life of the interior effect of the body strengthening substantially and increase.DNL ^tMother advantages of product also can comprise that lower immunogenicity, administration frequency reduce, dissolubility increases, stability strengthens and renal clearance reduces.

Background context

It is reported that interferon-' alpha ' (IFN α) is at animal cancer model (Ferrantini etc., 1994, J Immunol153:4604-15) and in human cancer patient (Gutterman etc., 1980, Ann Intern Med93:399-406) all there is anti-tumor activity.IFN α can bring into play multiple direct antitumor action, comprise downward oncogene, raise tumor-inhibiting factor, by increasing the expression of tumor surface MHC I albuminoid, strengthen immunity identification, reinforcement apoptosis, and the enhanced sensitivity (Gutterman etc. to chemotherapeutant, 1994, PNAS USA91:1198-205; Matarrese etc., 2002, Am J Pathol160:1507-20; Meechia etc., 2000, Gene Ther7:167-79; Sabaawy etc., 1999, Int J Oncol14:1143-51; Takaoka etc., 2003, Nature424:516-23).For some tumors, IFN α can by activation STAT1 have direct and effective antiproliferative effect (Grimley etc., 1998Blood91:3017-27).

Indirectly, IFN α can suppress angiogenesis (Sidky and Borden, 1987, Cancer Res47:5155-61) and stimulation of host immunocyte, it may react most important but be underestimated (Belardelli etc. to a great extent whole antitumor, 1996, Immunol Today17:369-72).IFN α passes through medullary cell (Raefsky etc., 1985, J Immunol135:2507-12; Luft etc., 1998, J Immunol161:1947-53), T cell (Carrero etc., 2006, J Exp Med203:933-40; Pilling etc., 1999, Eur J Immunol29:1041-50) and B cell (Le etc., 2001, effect Immunity14:461-70) and immunoreation is had to pleiotropy impact.As the important regulator of innate immune system, quick differentiation and activation (Belardelli etc., 2004, the Cancer Res64:6827-30 of IFN α induction dendritic cell; Paquette etc., 1998, J Leukoc Biol64:358-67; Santini etc., 2000, J Exp med191:1777-88) and strengthen that cytotoxicity, migration, cytokine generate and antibody dependent cellular cytotoxicity (ADCC) (Biron etc., 1999, the Annu Rev Immunol17:189-220 of NK cell; Brunda etc., 1984, Cancer Res44:597-601).

Up to now, by approval IFN-α 2, be used for the treatment of hairy cell leukemia, chronic granulocytic leukemia, malignant melanoma, follicular lymphoma, condyloma acuminatum, AID dependency Kaposi sarcoma (AIDs-related Kaposi sarcoma), chronic hepatitis B and hepatitis C; IFN-β is used for the treatment of multiple sclerosis; And IFN-γ is used for the treatment of chronic granulo matosis and pernicious osteopetrosis, the verified treatment effectiveness of IFN.Although the document about this group autocrine and the paracrine cell factor is numerous, its function in healthy and disease is still illustrated, and comprises the more effective and novel form (Pestka, 2007, the J.Biol.Chem282:20047-51 that introduce clinically; Vilcek, 2006, Immunity25:343-48).

Interferon plays a key effect in antitumor and antimicrobial host defense, and is extensively explored therapeutic agent (Billiau etc., 2006, the Cytokine Growth Factor Rev17:381-409 as cancer and infectious disease; Pestka etc., 2004, Immunol Rev202:8-32).Although I type and II type interferon (IFN-α/β and γ) have been made sizable effort, but the use due to its short circulating half-life, general toxicity and not good enough reaction in patient and in clinical configuration is limited (Pestka etc., 2004, Immunol Rev202:8-32; Miller etc., 2009, Ann N Y Acad Sci1182:69-79).At the beginning of 2003, the optional IFN agent of developing for these unsatisfied clinical indications that is found to be of IFN-λ family has brought-individual stem-winding new chance (Kotenko etc., 2003, Nat Immunol4:69-77; Sheppard etc., 2003, Nat Immunol4:63-8).

IFN-λ (being called type iii interferon) is the cytokine being comprised of IFN-λ 1,2,3 (being also called IF2 9,28A and 28B) of one group of up-to-date description, they carry out genetic coding (Kotenko etc. by being positioned at three different genes on chromosome 19,2003, Nat Immunol4:69-77; Sheppard etc., 2003, Nat Immunol4:63-8).On protein level, IFN-λ 2 and IFN-λ 3 height homologies, have 96% aminoacid homogeneity, and IFN-λ 1 and IFN-λ 2 and total approximately 81% homology (Sheppard etc., 2003, Nat Immunol4:63-8) of IFN-λ 3.IFN-λ by with by I type IFN, induced similar JAK/STAT pathway activation signal transduction, activation (the Witte etc. that comprise the phosphorylation of the kinase whose activation of JAK1 and TYK2, stat protein and the transcription complex of the gene factor 3 (ISGF3) that IFN stimulates, 2010, Cytokine Growth Factor Rev21:237-51; Zhou etc., 2007, J Virol81:7749-58).

Main Differences between III type IFN system and I type IFN system is the distribution of its receptor complex separately.IFN-α/β is carried out signal conduction by the I type interferon receptors of two kinds of wide expression, and use relevant gained general toxicity with IFN-α/β and limited them as the use (Pestka etc. of therapeutic agent, 2007, J Biol Chem282:20047-51).By contrast, the allos dimerization receptor complex that IFN-λ consists of the IFN-λ receptor 1 by unique (IFN-λ R1) and IL-10 receptor 2 (IL-10R2) carries out signal conduction.As previous, report (Witte etc., 2009, Genes Immun10:702-14), IFN-λ R1 has a very limited expression pattern, wherein in epithelial cell, melanocyte and hepatocyte, level is the highest, and level is minimum in primary central nervous system (CNS) cell.Haematogenic immunity system cells is expressed high-caliber short IFN-λ receptor splice variant (sIFN-λ R1), and it suppresses IFN-λ effect.The finite response of neuronal cell and immunocyte means, utilizes IFN-λ can make not exist to the frequent relevant serious toxicity of IFN-α therapy or significantly reduce (Witte etc., 2009, Genes Immun10:702-14; Witte etc., 2010, Cytokine Growth Factor Rev21:237-51).New publication report, although IFN-α and IFN-λ induce the expression of one group of common ISG (interferon-stimulated gene) in hepatocyte, but different from IFN-α, using not of IFN-λ can induce STAT activation or ISG to express (Dickensheets etc. in the lymphocyte of purification or mononuclear cell, 2013, J Leukoc Biol.93, is published in 12/20/12 on the net).This shows the treatment for chronic HCV infection, and IFN-λ may be better than IFN-α, because IFN-λ unlikely induces the often leukopenia (Dickensheets etc., 2013) relevant to IFN-α therapy.

IFN-λ shows the cytokine similar architectural feature relevant with IL-10, but in function, has I type IFN class antiviral and antiproliferative activity (Witte etc., 2009, Genes Immun10:702-14; Ank etc., 2006, J Virol80:4501-9; Robek etc., 2005, J Virol79:3851-4).The verified cytopathic effect that reduces virus replication or various viruses of IFN-λ 1 and IFN-λ 2, described virus comprises DNA viruses (hepatitis B virus (Robek etc., 2005, J Virol79:3851-4; Doyle etc., 2006,44:896-906) and herpes simplex types 2 virus (Ank etc., 2008, J Immunol180:2474-85)), ss (+) RNA viruses (EMCV; Sheppard etc., 2003, Nat Immunol4:63-8) and hepatitis C virus (Robek etc., 2005, J Virol79:3851-4; Doyle etc., 2006,44:896-906; Marcello etc., 2006, Gastroenterol131:1887-98; Pagliaccetti etc., 2008, J Biol Chem283:30079-89), ss (-) RNA viruses (vesicular stomatitis virus; Pagliaccetti etc., 2008, J Biol Chem283:30079-89) and influenza A virus (Jewell etc., 2010, J Virol84:11515-22) and diplornavirus as rotavirus (Pott etc., 2011, PNAS USA108:7944049).IFN-λ 3 is accredited as the key cytokines (Ge etc. in HCV infection according to genetic research, 2009, Nature461:399-401), and the lateral reactivity (Dellgren etc. of show needle to EMCV, 2009, Genes Immun10:125-31).Order of severity height correlation (the Contoli etc. that the shortage that the IFN-λ of rhinovirus induction produces it is reported the asthma aggravation brought out with rhinovirus, 2006, Nature Med12:1023-26) and IFN-lambda therapy be proposed as treatment allergic asthma new method (Edwards and Johnston, 2011, EMBO Mol Med3:306-8; Koltsida etc., 2011, EMBO Mol Med3:348-61).

In several human cancer cell line, determined the antiproliferative activity of IFN-λ, comprise neuroendocrine carcinoma BON1 (Zitzmann etc., 2006,344:1334-41), glioblastoma multiforme LN319 (Meager etc., 2005, Cytokine31:109-18), immortalization keratinocyte HaCaT (Maher etc., 2008, Cancer Biol Ther7:1109-15), melanoma F01 (Guenterberg etc., 2010, Mol Cancer Ther9:510-20) and esophageal carcinoma TE-11 (Li etc., 2010, Eur J Cancer46:180-90).In animal model, IFN-λ is by congenital and the reaction induced apoptosis of tumor cells of adaptive immunity and destruction, this shows that the local delivery of IFN-λ may be the useful auxiliary strategy (Numasaki etc., 2007, J Immunol178:5086-98) in human malignancies treatment.

In clinical configuration, Pegylation IFN-λ 1 (PEG-IFN-λ 1) by interim for thering is the patient of chronic hcv infection.In the Ib stage, study in (n=56), under all dosage levels (0.5-3.0 μ g/kg), all observe antiviral activity, and when PEG-IFN-λ 1 being applied to the gene 1 type HCV patient of recurring after IFN-α therapy, virus load is down to 4.0log (Muir etc. from 2.3,2010, Hepatology52:822-32).The IIb stage is studied (n=526) and shows that the patient with gene 1 type and 4 type HCV is for having significantly higher response rate with PEG-IFN-λ 1 treatment than PEG-IFN-α treatment.Meanwhile, relevant adverse events incidence rate is lower conventionally to I type interferon therapy than utilizing in the situation of PEG-IFN-α for the situation of utilizing PEG-IFN-λ 1.Seldom observe neutrophilic granulocyte and reduce and thrombocytopenia, and the incidence rate of influenza-like symptom, anemia and musculoskeletal symptom is down to PEG-IFN-α treatment being seen approximately 1/3.Yet the serious adverse events between PEG-IFN-λ 1 and PEG-IFN-α, depression and other common adverse events (>=10%) incidence rates are similar.Compare with PEG-IFN-α, the visible more liver toxicity of height ratio (" Investigational Compound PEG-Interferon Lambda Achieved Higher Response Rates with Fewer Flu-like and Musculoskeletal Symptoms and Cytopenias Than PEG-Interferon Alfa in Phase IIb Study of526Treatment-Naive Hepatitis C Patients in maximum dose level PEG-IFN-λ 1, " on April 2nd, 2011, Press Release from Bristol-Myers Squibb).

Compositions and the method that need to comprise interferon-λ-antibody complex, described complex keeps the biological activity of unmodified interferon, but shows toxicity and/or the excellent pharmacokinetic property of effect, reduction in improved body.

Summary of the invention

The invention discloses for generation of DNL ^tMthe method and composition of complex, described complex comprises and is connected to the interferon of antibody or antigen binding antibody fragment, preferably interferon-λ, more preferably IFN-λ 1.Described interferon part can be bonded to anchoring structure territory (AD) part that human kinase protein A (PKA) regulates the dimerization of subunit RI α, RI β, RII α or RII β and stops domain (DDD) partly or yoke is bonded to A kinases anchorin (AKAP) alternatively by yoke.Antibody or antibody fragment conjugated part are bonded to complementary AD or DDD part.Because DDD part spontaneously forms dimer, described dimer is incorporated into AD part with high-affinity, so can be by the dress such as the effect subgroup such as interferon, antibody or antibody fragment DNL ^tMcomplex, described effector is connected to DDD part or AD part separately.By the effector that is connected to DDD part is mixed with the effector that is connected to AD part, any in fact effector can be incorporated to DNL ^tMin complex.

DNL ^tMcomplex preferably contains one, the interferon part that is connected to antibody or fragment of two or four copy.Yet, those skilled in the art will recognize that, can in the scope of method and composition required for protection, build and use the DNL of the other types with different structures and different interferon/antibody ratio ^tMcomplex, for example U.S. Patent No. 7,521, disclosed in 056,7,527,787,7,534,866,7,550,143 and 7,666,400.In a more preferred embodiment, can the appropriate location by DDD and AD sequence introduce cysteine residues form can stabilisation complex disulfide bond, thereby by DNL ^tMcomplex covalence stablility.

In other preferred embodiments, comprise the DNL that interferon yoke closes antibody ^tMcomplex shows the serum clearance rate that closes slow at least one order of magnitude of interferon than yoke not.

Those skilled in the art will recognize that, this technology is not limited to interferon-λ, but can be applicable to the targeted delivery of multiple therapeutic agent, described therapeutic agent includes, but is not limited to enzyme, cytokine, chemotactic factor, somatomedin, peptide, fit, hemoglobin, antibody and its fragment.Exemplary dose comprises MIF, HMGB-1 (high mobility group protein 1), TNF-α, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-19, IL-21, IL-23, IL-24, CCL19, CCL21, IL-8, MCP-1, RANTES, MIP-1A, MIP-1B, ENA-78, MCP-1, IP-10, Gro-β, eotaxin (Eotaxin), interferon-' alpha ', interferon-beta, interferon-γ, interferon-λ, G-CSF, GM-CSF, SCF, PDGF, MSF, Flt-3 part, erythropoietin (erythropoietin), thrombopoietin (thrombopoietin), hGH, CNTF, leptin (leptin), oncostatin M, VEGF, EGF, FGF, PIGF, insulin, hGH, calcitonin, Factor IX, IGF, somatostatin, tissue plasminogen activator and LIF.

The antibody that can be used for the targeting cancer therapy in the scope of method and composition required for protection includes, but is not limited to LL1 (anti-CD74), LL2 and RFB4 (anti-CD22), RS7 (anti-Glycoproteins in Epithelial-1 (EGP-1)), PAM4 and KC4 (being all anti-stick albumen), MN-14 (anticancer embryonal antigen (CEA, also referred to as CD66e), MN-15 (anti-CEACAM6), Mu-9 (anti-colon-specific antigen-p), Immu-31 (Anti-α-Fetoprotein), anti-TAG-72 (for example CC49), anti-Tn, J591 or HuJ591 (anti-PSMA (prostate specific membrane antigen)), AB-PG1-XG1-026 (anti-PSMA dimer), D2/B (anti-PSMA), G250 (anti-carbonic anhydrase IX), hL243 (anti-HLA-DR), alemtuzumab (alemtuzumab) (anti-CD52), bevacizumab (bevacizumab) (anti-VEGF), Cetuximab (cetuximab) (anti-EGFR), lucky trastuzumab (gemtuzumab) (anti-CD 33), ibritumomab tiuxetan (ibritumomab tiuxetan) (anti-CD20), Victibix (panitumumab) (anti-EGFR), Rituximab (rituximab) (anti-CD20), tositumomab (tositumomab) (anti-CD20), GA101 (anti-CD20), and Herceptin (trastuzumab) (anti-ErbB).These antibody are (for example, U.S. Patent No. 5,686,072,5,874,540,6,107,090,6 as known in the art, 183,744,6,306,393,6,653,104,6,730.300,6,899,864,6,926,893,6,962,702,7,074,403,7,230,084,7,238,785,7,238,786,7,256,004,7,282,567,7,300,655,7,312,318,7,585,491,7,612,180,7,642,239; With U.S. Patent Application Publication No.20040202666 (now waiving the right), 20050271671 and 20060193865; The embodiment of each document is partly incorporated herein by reference).Concrete known antibodies used comprises hPAM4 (U.S. Patent No. 7, 282, 567), hA20 (U.S. Patent No. 7, 251, 164), hA19 (U.S. Patent No. 7, 109, 304), hIMMU31 (U.S. Patent No. 7, 300, 655), hLL1 (U.S. Patent No. 7, 312, 318), hLL2 (U.S. Patent No. 7, 074, 403), hMu-9 (U.S. Patent No. 7, 387, 773), hL243 (U.S. Patent No. 7, 612, 180), hMN-14 (U.S. Patent No. 6, 676, 924), hMN-15 (U.S. Patent No. 7, 541, 440), hR1 (U.S. Patent application 12/772, 645), hRS7 (U.S. Patent No. 7, 238, 785), hMN-3 (U.S. Patent No. 7, 541, 440), AB-PG1-XG1-026 (U.S. Patent application 11/983, 372, with ATCC PTA-4405 and PTA-4406, deposit) and D2/B (WO2009/130575), described in each, the text of patent or application is incorporated herein in the mode of partly quoting about drawings and Examples.

Anti-TNF-α antibody is as known in the art and can be used for treating immunological diseases, such as asthma (referring to such as Erin etc., 2006, Am J Respir Crit Care Med174:753-62).The known antibody for TNF-α comprise human antibodies CDP571 (Ofei etc., 2011, Diabetes45:881-85); Rodent antibody MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI, M302B and M303 (Thermo Scientific, Rockford, IL); Infliximab (infliximab) (Centocor, Malvern, PA); Pegylation match trastuzumab (certolizumab pegol) (UCB, Brussels, Belgium); And adalimumab (adalimumab) (Abbott, Abbott Park, IL).These and many other known anti-TNF-α antibodies can be used in method and composition required for protection.Other antibody that use include, but is not limited to anti-B cell antibody, for example, tie up trastuzumab (veltuzumab), epratuzumab (epratuzumab), meter La Zhu monoclonal antibody (milatuzumab) or hL243; Holder pearl monoclonal antibody (tocilizumab) (anti-IL-6 receptor); Basiliximab (basiliximab) (anti-CD25); Daclizumab (daclizumab) (anti-CD25); Pearl monoclonal antibody (efalizumab) (anti-CD11a) in accordance with the law; Luo Dankang (muromonab)-CD3 (anti-CD3 receptor) not; Anti-CD 40 L (UCB, Brussels, Belgium); Natalizumab (natalizumab) (anti-α 4 integrins) and omalizumab (omalizumab) (anti-IgE).

Macrophage migration inhibitory factor (MIF) is congenital and adaptive immunity and apoptotic important regulatory factor.Reported that CD74 is the endogenous recipient (Leng etc., 2003, J Exp Med197:1467-76) of MIF.The anti-CD74 antibody of Antagonism can be used for treating extensive various disease states for the therapeutical effect of approach in the cell of MIF mediation, for example bladder cancer, carcinoma of prostate, breast carcinoma, pulmonary carcinoma, colon cancer and chronic lymphocytic leukemia are (for example, Meyer-Siegler etc., 2004, BMC Cancer12:34; Shachar and Haran, 2011, Leuk Lymphoma52:1446-54); Nephropathy, for example kidney allograft rejection reaction (Lan, 2008, Nephron Exp Nephrol.109:e79-83); And numerous inflammatory diseases (electronic publishing was on March 22nd, 2009 for Meyer-Siegler etc., 2009, Mediators Inflamm; Takahashi etc., 2009, Respir Res10:33).Meter La Zhu monoclonal antibody (hLL1) is the exemplary anti-CD74 antibody with the therapeutic use that is used for the treatment of Jie's MIF mediated diseases.

Pharmaceutical composition of the present invention can be used for treatment and has neurodegenerative disease as the experimenter of Alzheimer.A bar pearl monoclonal antibody (bapineuzumab) is in the clinical trial for the treatment of of alzheimer's disease.Other antibody that are proposed to be used in treatment of alzheimer's disease comprise Alz50 (Ksiezak-Reding etc., 1987, J Biol Chem263:7943-47), more spit of fland reed monoclonal antibody (gantenerumab) and Su Lan pearl monoclonal antibody (solanezumab).Infliximab (a kind of anti-TNF-α antibody) has been in the news and can have reduced amyloid speckle and improve cognitive.In the neurofibrillary tangles of Alzheimer brain in patients there is (Bryan etc., Mol Neurodegeneration2008 in CD74's; 3:13doi:10.1186/1750-1326-3-13) show that CD74 can be the target of peptide or antibody therapy, or for making these regions of therapeutic agent targeting Alzheimer brain in patients.

Spendable other antibody comprise for infectious disease pathogen as the antibody of antibacterial, virus, mycoplasma or other pathogen.The much antibody for these infectious pathogens is that as known in the art and any this known antibody all can be used in method and composition required for protection.For example, for the antibody of the gp120 glycoprotein antigen of HIV (human immunodeficiency virus) I (HIV-1), be known, and some this antibody can have immanoprotection action in the mankind.Referring to such as Rossi etc., Proc.Natl.Acad.Sci.USA.86:8055-8058,1990.Known anti-HIV antibody comprises (AIDS.2006 October 3 by Johansson etc.; 20 (15): the anti-peplos antibody of 1911-5) describing, and the anti-HIV antibody of describing and selling by Polymun (Vienna, Austria), it is also described in United States Patent (USP) 5,831,034, United States Patent (USP) 5,911,989, and Vcelar etc., AIDS2007; 21 (16): 2161-2170 and Joos etc., Antimicrob.Agents Chemother.2006; 50 (5): in 1773-9, be all incorporated herein by reference.

Antibody for malarial parasite can be for zygoblast, merozoite, schizont and gametocyte stage.Generated the monoclonal antibody for zygoblast (Circumsporozoite antigen), and shown its in vitro with rodent in can in and zygoblast (N.Yoshida etc., Science207:71-73,1980).Some team have developed toxoplasma antibody, and toxoplasma is protozoon parasite (Kasper etc., J.Immunol.129:1694-1699,1982 related in toxoplasmosis; The same, 30:2407-2412,1983).Developed for the antibody of virgin worm (schistosomulae) surface antigen and found it in vivo or interaction in vitro in virgin worm (Simpson etc., Parasitology, 83:163-177,1981; Smith etc., Parasitology, 84:83-91,1982; Gryzch etc., J.Immunol., 129:2739-2743,1982; Zodda etc., J.Immunol.129:2326-2328,1982; Dissous etc., J.Immunol., 129:2232-2234,1982).

Schizotrypanum cruzi (Trypanosoma cruzi) is the pathogen of American trypanosomiasis (Chagas ' disease), and hunts stinkbug entomochory by what suck blood.Generate specificity in vitro and suppressed parasitic a kind of form to the differentiation of another kind of form (epimastigote is to the trypomastigote stage) and the antibody reacting with cell surface glycoprotein; Yet there is not this antigen (Sher etc., Nature, 300:639-640,1982) in parasitic mammal (blood flow) form.

Anti fungal antibody is as known in the art, for example anti-sclerotiniose antibody (United States Patent (USP) 7,910,702); Anti-glucuronoxylose mannan (antiglucuronoxylomannan) antibody (Zhong and Priofski, 1998, Clin Diag Lab Immunol5:58-64); Anti-candida belong to antibody (Matthews and Burnie, 2001,2:472-76); And anti-glycosyl sphingolipid antibody (Toledo etc., 2010, BMC Microbiol10:47).

Developed the suitable antibodies that causes most of microorganism (antibacterial, virus, protozoacide, fungus, other parasites) of infecting for great majority in the mankind, and many previously for in-vitro diagnosis object.The antibody for extensive multiple pathogens of commercially available acquisition is known and can be utilized.Exemplary antipathogen antibody from Millipore (Billerica, MA) comprises Chinese People's Anti-Japanese Military and Political College enterobacteria (E.coli) antibody (MAB8272), anti-salmonella belongs to (Salmonella) antibody (MAB748-MG-K), anti-listeria monocytogenes (Listeria monocytogenes) antibody (MAB8001), anti-helicobacter pylori (Helicobacter pylori) antibody (IHC2140-6), anti-Staphylococcus aureus (Staphylococcus aureus) antibody (MAB930), Against Chlamydia Trachomatis (Chlamydia trachomatis) antibody (AB1120F), anti-Mycoplasma bovis (Mycoplasma bovis) antibody (MAB970), anti-Mus giardia lamblia stiles (Giardia muris) antibody (MAB10242), anti-dengue virus (Dengue virus) antibody (MAB10226), anti-flavivirus belongs to (Flavivirus) antibody (MAB10216), anti-legionella pneumophilia (L.pneumophila) antibody (MAB10223), resistance of hepatitis B antibody (MAB10201), anti-dengue virus antibody (MAB10217), anti-herpes simplex antibody (MAB8685), anti-Epstein epstein-Barr virus (Epstein Barr virus) antibody (MAB10219), anti-Coxsackie virus (Coxsackie virus) antibody (MAB10220), preventing respiratory syncytial virus (Respiratory Syncytial virus) antibody (MAB8262-KC), Anti-adenovirus antibody (MAB8043-KC), antibodies against influenza virus (MAB8661-KC), anti-rsv antibodies (MAB8594), anti-papillomavirus (Papillomavirus) antibody (MAB837), anti-HCV (AB307), anti-enteric virus71 antibody (MAB979-K), anti-measles antibody (MAB8905-K), anti-cytomegalovirus (Cytomegalo virus) antibody (MAB8140-KC), anti-yellow fever virus (Yellow Fever virus) antibody (MAB984-K), anti-rabies antibody (MAB8724), anti-herpesvirus 6 antibody (MAB8535), poliomyelitis virus (Poliovirus) antibody (MAB8566), anti-SARS antibody (MAB8785), anti-new castle disease virus (Newcastle Disease virus) antibody (MAB80144) and anti-west Nile virus (West Nile virus) antibody (MAB8150).From Santa Cruz Biotechnology (Santa Cruz, CA) exemplary antipathogen antibody comprises anti-dengue virus antibody (sc-325018), anti-vaccinia virus antibody (sc-69949), anti-polyoma virus antibody (sc065925), rubella virus antibody (sc101364), anti-Ebola virus (Ebola virus) antibody (sc-51872), anti-EBV antibody (sc-17500), anti-measles antibody (sc-58167), material for anti parotitis antibody (sc-57918), anti-hantavirus (Hantavirus) antibody (sc-57755) and anti-mycoplasma hominis (Mycoplasma hominis) virus (sc-58171).Exemplary antipathogen antibody from ProSci Inc. (Poway, CA) comprises anti-Eurotium (Aspergillus) antibody (35-595), anti-candida albicans (Candida albicans) antibody (35-121) and anti-saccharomyces cerevisiae (Saccharomyces cerevisiae) antibody (35-361).Exemplary antipathogen antibody from KPL (Gaithersburg, MD) comprises that anti-Staphylococcus aureus antibody (01-90-05), anti-lyme disease spirochete (Borrelia burgdorferi) antibody (01-97-91), anti-pylori spiral bacilli antibody (01-93-94), anti-Legionella belong to (Legionella spp.) antibody (01-90-03) and anti-yersinia's genus (Yersinia spp.) antibody (01-90-04).There is the business antipathogen antibody in many other sources well known in the art.Those skilled in the art will recognize that the commercially available acquisition of antibody and/or open acquisition for any in fact interested pathogen, and any this type of known antibody can be incorporated to the DNL of use method and composition hereinafter described ^tMin complex.

Described DNL ^tMcomplex is applicable in extensive multiple treatment and diagnostic application.DNL ^tMthe using method of complex can comprise detection, diagnosis and/or treatment disease or other medical science condition of illness.These condition of illness can include, but is not limited to cancer, hypertrophy, asthma, multiple sclerosis, infectious disease, chronic viral hepatitis, herpesvirus infection, chronic type b or infection with hepatitis C virus, chronic granulo matosis, pernicious osteopetrosis, Kaposi sarcoma (Karposi ' s sarcoma), human papilloma virus infection, influenza, chronic granulocytic leukemia, hairy cell leukemia, cutaneous T cell lymphoma, follicular lymphoma, metastatic renal cell cancer, hemangioma, hematology's malignant tumor, condyloma acuminatum and malignant melanoma.

Medicable exemplary oncologic type comprises acute lymphoblastic leukemia, acute myeloid leukemia, gallbladder cancer, breast carcinoma, cervical cancer, chronic lymphocytic leukemia, chronic granulocytic leukemia, colorectal carcinoma, carcinoma of endometrium, esophageal carcinoma, gastric cancer, head and neck cancer, Hodgkin lymphoma (Hodgkin ' s lymphoma), pulmonary carcinoma, medullary thyroid carcinoma, non-Hodgkin lymphoma, multiple myeloma, renal carcinoma, ovarian cancer, cancer of pancreas, glioma, melanoma, hepatocarcinoma, carcinoma of prostate and bladder cancer.

Can treat various viral infection, include, but is not limited to HIV (human immunodeficiency virus) (HIV), herpesvirus, herpes simplex virus, vaccinia virus, cytomegalovirus, rabies virus, influenza virus, rhinovirus, hepatitis B virus, hepatitis C virus, Sendai virus (Sendai virus), feline leukaemia virus, reovirus (Reo virus), poliovirus, the tiny sample virus of serum human, simian virus 40, respiratory syncytial virus, mouse mammary tumor virus, varicella zoster virus, dengue virus, rubella virus, Measles virus, adenovirus, human T-leukemia virus, epstein-Barr virus, muroid leucovirus, mumps virus, vesicular stomatitis virus, sindbis alphavirus (Sindbis virus), lymphocytic choriomeningitis virus, Verrucosis poison and blue tongue rims, or the group forming below antibacterial choosing freely: streptococcus agalactiae (Streptococcus agalactiae), legionella pneumophilia (Legionella pneumophila), micrococcus scarlatinae (Streptococcus pyogenes), escherichia coli (Escherichia coli), Diplococcus gonorrhoeae (Neisseria gonorrhoeae), Neisseria meningitidis (Neisseria meningitidis), streptococcus pneumoniae (Pneumococcus), influenza B haemophilus (Hemophilus influenzae B), syphilis spirillum (Treponema pallidum), lyme disease spirochete (Lyme disease spirochetes), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Mycobacterium leprae (Mycobacterium leprae), Bacillus abortus (Brucella abortus), mycobacterium tuberculosis (Mycobacterium tuberculosis) or clostridium tetani (Clostridium tetani).

Brief Description Of Drawings

Fig. 1 is the cartoon figure of gene structure (A and B) that describes the expression of cytokine-DDD2 (C) and IgG-AD2 (D) DNL module.Described module is combined to form the DNL structure forming to four cytokines of IgG (E) by merging.

Fig. 2 shows the external IFN alpha active that cytokine-MAb DNL construct is compared with Pegylation or primary IFN α.Measurement activity specific as be shown in the examples (IU/pmol).Activity from each test article of rhIFN α 2b standard curve extrapolation concentration known.At 20-2b (●), the 734-2b of progressive concentration (■), v-mab (zero), v-mab+734-2b (), PEGASYS , PEG-intron (▲) or 1R-2b under existence, make culture growth and measure relative viable cell density with MTS.The % of the signal that drafting obtains from untreated cell is with respect to the curve of the log value of molar concentration.Use Prism Software Create dose-response curve and EC ₅₀value.Error bars, SD.

The reporter gene algoscopy of Fig. 2 (A) based on cell.

Fig. 2 (B) utilizes the virus protection algoscopy of EMC virus and A549 cell.

Fig. 2 (C) is used the external lymphoma proliferation assay of Daudi cell.

Fig. 2 (D) is used the external lymphoma proliferation assay of Jeko-1 cell.

Fig. 3 shows the pharmacokinetic analysis result in Swiss-Webster mice.The IFN α 2b concentration of using 20-2b, α 2b-413, PEG intron or PEGASYS and passing through in elisa assay blood serum sample through 96 hours to mice.Show serum and eliminate curve.Serum half-life (T _1/2) eliminate speed and mean residence time (MRT) is summarized in the form of insertion.

Fig. 4 (A) shows the ADCC effector function of 20-2b.Under the PBMC of fresh separated exists by incubation together with 20-2b, 22-2b, v-mab, epratuzumab (e-mab) or the h734 of Daudi or Raji cell and 5 μ g/ml 4 hours, quantitative cytolysis subsequently.

Fig. 4 (B) shows the CDC effector function of 20-2B.Under human complement exists by the serial dilution of Daudi cell and 20-2b (●), 734-2b (■) or v-mab (zero) together incubation.Draw complement control % (the living cells number in test sample, than the cell of only being processed by complement) with respect to the curve of the log value of nM concentration.Error bars, SD.

Fig. 5 shows 20-2b to be strengthened the consumption of the NHL cell from whole blood.Fresh heparinization human blood is mixed with Daudi or Ramos and with 0.01,0.1 or 20-2b (●), v-mab (zero), 734-2b (■) or the v-mab+734-2b () of 1nM incubation two days together.Use the impact for the treatment of on lymphoma and peripheral blood lymphocyte shown in the evaluation of fluidic cell surveying.Error bars, SD.

Fig. 6 (A) shows the survival curve that shows the therapeutic efficiency of 20-2b in dissemination Burkitt lymphoma (Daudi) xenograft models.At the 0th day, in female C.B.17SCID mouse vein, use Daudi cell.Treatment is by 20-2b (●), the 734-2b (■) that provide with single subcutaneous dosage, v-mab (zero), PEGASYS or saline (X) forms.Treatment natural law is indicated with arrow.Use Prism software analysis survival curve.In early days in Daudi model, the 1st day group to 10 mices, provide the single dose of 0.7pmol (solid line) or 0.07pmol (dotted line).

Fig. 6 (B) shows with Fig. 6 (A) and similarly studies, but late in Daudi model.The single dose of 0.7pmol (solid line), 7pmol (dotted line) or 70pmol (gray line) is provided the 7th day group to 10 mices.

Fig. 7 (A) has presented the survival curve of the therapeutic efficiency of demonstration 20-2b in dissemination Burkitt lymphoma (Raji and NAMALWA) xenograft models.At the 0th day, in female C.B.17SCID mouse vein, use NHL cell.Treatment is comprised of 20-2b (●), the 734-2b (■) that provide with subcutaneous dosage, v-mab (zero) or saline (X).Treatment natural law is indicated with arrow.Use Prism software analysis survival curve.Late in Raji model, in the groups of 10 mices of the 5th, 7,9,12,14 and 16 angels, accept 250pmol dosage.

Fig. 7 (B) shows with Fig. 7 (A) and similarly studies, but in early days in NAMALWA model.The group of 6 mices accepted 20-2b or the 734-2b of 250pmol dosage or at the 1st, 5,9,13,17,21 and 25 days, accepts the v-mab of 3.5nmol dosage at the 1st, 3,5,8,10 and 12 days.

The schematic diagram that Fig. 8 (A) AD2-IFN-λ 1 expresses module.Figure discloses respectively SEQ ID NO99-100 by outward appearance order.(B) show interferon-antibody DNL ^tMthe schematic diagram of the structure of module.

The cell surface expression of Fig. 9 antigen in various cell line.

Figure 10 (A) (E1)-λ 1 and ME-180 cell, (B) (15)-λ 1 and HepG2 cell and (C) (C2)-1 strengthen to some extent than AD2-λ 1 module with the combination activity of A375 cell.

Figure 11 (E1)-λ 1 is for the cytotoxic effect of ME-180 cell.

Figure 12 (15)-λ 1 is for the cytotoxic effect of ME-180 cell.

Figure 13. antivirus action.(A) the anti-HCV effect of (c225)-λ 1 in Huh-7 cell strengthens.With shown in (c225)-λ 1, (the C2)-λ 1 of concentration or 1 dose of processing of rhIFN-λ there is the Huh-7 stable cell lines of the HCV genotype 1 b Con1 replicon of expressing LUC Photinus pyralis LUC Photinus pyralis FL.After 3 days, measure uciferase activity and pass through the activity decreased percentage test antivirus action with respect to untreated cell.Utilize Graph Pad Prism to use S shape matching (variable slope) to carry out analytical data.Sample is twice of independent operation in duplicate.(B) the anti-EMCV effect of (15)-λ 1 in A549 cell strengthens.Before exciting with EMCV, make incubation together with the serial dilution of A549 cell and (15)-λ 1, (C2)-λ 1, rhIFN-λ 1 or hMN15-Fab-DDD2.Estimate cytopathic effect (CPE) and measure, and utilize GraphPad Prism to use S shape matching (variable slope) to carry out analytical data.Sample is twice of independent operation in duplicate.

Describe in detail

Definition

Unless otherwise indicated, otherwise " one " means " one or more ".

As used herein, term " with " can be used for referring to conjunction or extract with "or".That is, except as otherwise noted, otherwise these two terms are interpreted as being equal to "and/or".

" therapeutic agent " is atom, molecule or the compound that can be used for treating disease.The example of therapeutic agent comprises antibody, antibody fragment, peptide, medicine, toxin, enzyme, nuclease, hormone, immunomodulator, antisense oligonucleotide, siRNA (siRNA), chelating agen, boron compound, photosensitizer, dyestuff and radiosiotope.

" diagnostic agent " is atom, molecule or the compound that can be used for diagnosing the illness.Useful diagnostic agent includes, but is not limited to radiosiotope, dyestuff (for example, together with biotin-Streptavidin complex), contrast agent, fluorescent chemicals or molecule, and for example, for the reinforcing agent (, paramagnetic ion) of nuclear magnetic resonance (MRI).

" antibody " refers to total length (be naturally occurring or form by normal immunoglobulin gene fragment recombination method) immunoglobulin molecules (for example IgG antibody) as used herein, or a part with the immunoglobulin molecules of immunocompetence (being specific binding), as antibody fragment." antibody " comprises monoclonal antibody, polyclonal antibody, bi-specific antibody, multi-specificity antibody, rodent antibody, chimeric antibody, humanized antibody and human antibodies.

" naked antibody " is antibody or its Fab that is not connected to therapeutic agent or diagnostic agent.The Fc part of complete naked antibody can provide effector function, for example complement fixation and ADCC (referring to for example Markrides, Pharmacol Rev50:59-87,1998).Other mechanism of naked antibody induction cell death can comprise apoptosis.(Vaswani and Hamilton, Ann Allergy Asthma Immunol81:105-119,1998.)

" antibody fragment " is a part for complete antibody, for example F (ab ') ₂, F (ab) ₂, Fab ', Fab, Fv, sFv, scFv, dAb etc.Regardless of structure, the same antigen that antibody fragment is identified with full length antibody is combined.For example, antibody fragment comprises the isolated fragment being comprised of variable region, " Fv " fragment being for example comprised of the variable region of heavy chain and light chain or the recombinant single chain peptide molecule (" scFv albumen ") that is connected light chain and variable region of heavy chain by peptide linker." single-chain antibody " (being often abbreviated as " scFv ") is by comprising V _hand V _lthe polypeptide chain of domain forms, and described domain interaction is to form antigen binding site.V _hand V _ldomain is connected by the peptide with 1 to 25 amino acid residue conventionally.Antibody fragment also comprises double-chain antibody, three chain antibodies and single domain antibody (dAb).

If it is upper significant that the amount of application of antibody or immune conjugate preparation or compositions as herein described is physiology, be referred to as with " treatment effective dose " and use.If the existence of medicament causes that detectable variation occurs acceptor experimenter's physiology, described medicament is for significant on physiology.In specific embodiment, if having caused antitumor, the existence of antibody preparation reacts or alleviated sign and the symptom of morbid state, described antibody preparation is for significant on physiology.The upper significant effect of physiology can also be body fluid and/or the cell immune response that has caused acceptor experimenter, causes target cell growth to be suppressed or death.

DOCK-AND-LOCK ^TM (DNL ^TM )

In preferred embodiments, interferon-antibody complex is with DOCK-AND-LOCK ^tM(DNL ^tM) composite form formation (referring to for example U.S. Patent No. 7,521,056,7,527,787,7,534,866,7,550,143 and 7,666,400, its embodiment separately is partly incorporated herein by reference).In general, described technology utilization occur in cAMP deopendent protein kinase (PKA) adjusting (R) subunit dimerization and stop domain (DDD) sequence and be derived from specificity and high-affinity binding interactions (Baillie etc., the FEBS Letters.2005 between any one anchoring structure territory (AD) sequence in multiple AKAP albumen; 579:3264; Wong and Scott, Nat.Rev.Mol.Cell Biol.2004; 5:959).DDD and AD peptide can be connected to any protein, peptide or other molecules.Because DDD sequence is dimerization be incorporated into AD sequence spontaneously, so described technology allows to form complex between any selected molecule that can be connected to DDD or AD sequence.

Although the DNL of standard ^tMcomplex comprises and has two DDD and connect molecules and be connected to the trimer that an AD connects molecule, but the variation of composite structure allows to form dimer, trimer, the tetramer, pentamer, six aggressiveness and other polymers.In some embodiments, DNL ^tMcomplex can comprise two or more antibody, antibody fragment or fusion rotein, and it is incorporated into identical antigenic determinant or is incorporated into two or more different antigen.DNL ^tMcomplex also can comprise one or more other effectors, for example protein, peptide, immunomodulator, cytokine, interleukin, interferon, in conjunction with albumen, peptide part, carrier protein, toxin, ribonuclease if ranpirnase (onconase), inhibition oligonucleotide are if siRNA, antigen or heteroantigen, polymer are as PEG, enzyme, therapeutic agent, hormone, cytotoxic agent, anti-angiogenic agent, short apoptosis agent or any other molecule or aggregation.

In the signal transduction pathway of the best research that PKA triggers in the combination by second message,second messenger cAMP and R subunit, play a key effect, first it separate (Walsh etc., J.Biol.Chem.1968 in nineteen sixty-eight from rabbit skeletal muscle; 243:3763).The structure of holoenzyme forms (Taylor, J.Biol.Chem.1989 by two catalytic subunits that remain inactive form by R subunit; 264:8443).The isozyme of finding PKA has the R subunit (RI and RII) of two types, and each type has α and β isoform (Scott, Pharmacol.Ther.1991; 50:123).Therefore, four kinds of isoforms of PKA adjusting subunit are RI α, RI β, RII α and RII β.Described R subunit has only shown that with stable dimeric forms separation and dimerization territory front 44 n terminal residues by RII α form (Newlon etc., Nat.Struct.Biol.1999; 6:222).As discussed below, other regulate the similar portions of the aminoacid sequence of subunit to be involved in dimerization and stop, are positioned at separately near the N-terminal that regulates subunit.The combination of cAMP and R subunit causes the release of the wide spectrum activity of serine/threonine kinases of active catalytic subunit, and described subunit is stopped together with AKAP by it PKA is divided and is positioned selected substrate (Scott etc., J.Biol.Chem.1990; 265; 21561).

Since first AKAP since to be microtubule-associated protein-2 characterized in 1984 (Lohmann etc., Proc.Natl.Acad.Sci US A.1984; 81:6723), in scope, in the species from yeast to the mankind, identified the more than 50 kinds AKAP that structure is various, it is positioned various Subcellular locations, comprise plasma membrane, actin cytoskeleton, nucleus, mitochondrion and endoplasmic reticulum (Wong and Scott, Nat.Rev.Mol.Cell Biol.2004; 5:959).The AD that is used for the AKAP of PKA is amphipathic helix (Carr etc., the J.Biol.Chem.1991 with 14-18 residue; 266:14188).The aminoacid sequence of AD between independent AKAP is very different, wherein reports that for the dimeric binding affinity scope of RII be 2 to 90nM (Alto etc., Proc.Natl.Acad.Sci.USA.2003; 100:4445).AKAP will only be incorporated into dimerization R subunit.For mankind RII α, AD is incorporated into hydrophobic surface (Colledge and Scott, the Trends Cell Biol.1999 being formed by 23 n terminal residues; 6:216).Therefore, the dimerization domain of mankind RII α and AKAP are positioned (Newlon etc., Nat.Struct.Biol.1999 in identical 44 amino acid whose sequences of N end in conjunction with territory; 6:222; Newlon etc., EMBO is J.2001; 20:1651), it is referred to herein as DDD.

We have developed following platform technology, wherein utilize mankind PKA to regulate the DDD of subunit and the AD of AKAP as a pair of outstanding joint module, being used for stopping any two entities (hereinafter referred to as A and B) becomes non-covalent complex, and it can further lock and become DNL to promote disulfide bond to form by introducing cysteine residues in the strategic position of DDD and AD ^tMcomplex.The conventional method opinion of described method is as follows.Entity A, by DDD sequence being connected to the precursor of A and building, obtains first ingredient, thus hereinafter referred to as a.Because described DDD sequence will realize dimeric spontaneous formation, so A will be by a ₂form.Entity B is to build by AD sequence being connected to the precursor of B, obtains thus second ingredient, is referred to as b.A ₂in the dimerization motif of contained DDD AD sequence that generation is used for comprising in the b stop site of being combined, thereby promote a ₂associate to form by a with the preparation of b ₂the trimerization complex of the binary that b forms.This binding events is because the reaction of covalently fixing two entities by disulfide bond bridge subsequently becomes irreversible, due to initial binding interactions should make to be positioned at reactive mercapto locus specificity on DDD and AD close (Chmura etc., Proc.Natl.Acad.Sci.USA.2001; 98:8480) to connect, according to the principle of effectiveness local concentration, this reaction will efficiently occur.Use the various combinations of joint, fit module and precursor, can produce and use the extensively multiple DNL with different chemical metering ^tMconstruct (referring to for example U.S. Patent No. 7,550,143,7,521,056,7,534,866,7,527,787 and 7,666,400).

By DDD is connected away from the functional groups of two kinds of precursors with AD, these locus specificities connect the original activity that also expection retains these two kinds of precursors.This method is essentially modular and potential locus specificity and the material that is covalently connected broad range of can be used for, and comprises peptide, protein, antibody, antibody fragment and has other active effector parts of broad range.Utilize the fusion rotein method that below the structure AD described in embodiment and DDD yoke close effector, any in fact protein or peptide can be incorporated to DNL ^tMin construct.Yet this technology is unrestricted and can utilize other yokes to close method.

Become known for manufacturing the several different methods of fusion rotein, comprise that nucleic acid is synthetic, hybridize and/or increase to produce the synthetic double-strandednucleic acid of the interested fusion rotein of coding.This type of double-strandednucleic acid can insert in expression vector, for produce fusion rotein (referring to such as Sambrook etc., Molecular Cloning, A laboratory manual, the 2nd edition, 1989) by standard molecular biological technique.In these preferred embodiments, AD and/or DDD partly can be connected to N-terminal or the C-terminal of effect protein or peptide.Yet, those skilled in the art will recognize that, according to relating to the part of its physiologically active in the chemical property of effector part and effector part, the site that connects AD or DDD part in effector part may be different.Can come locus specificity to connect multiple effector part by technology known in the art, for example, use bivalent cross-linking reagent and/or other chemical yokes to close technology.

structure-function relationship in AD and DDD part

For dissimilar DNL ^tMconstruct, can utilize different AD or DDD sequence.Exemplary DDD and AD sequence are below provided.

DDD1

SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：1)

DDD2

CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：2)

AD1

QIEYLAKQIVDNAIQQA(SEQ?ID?NO：3)

AD2

CGQIEYLAKQIVDNAIQQAGC(SEQ?ID?NO：4)

Those skilled in the art will recognize that, DDD1 and DDD2 are the DDD sequences of the mankind RII α isoform based on protein kinase A.Yet in optional embodiment, DDD sequence and the corresponding AKAP sequence of the protein kinase A that DDD and AD part may be based on mankind RI alpha forms, as illustrated in below DDD3, DDD3C and AD3.

DDD3

SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK(SEQ?ID?NO：5)

DDD3C

MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK(SEQ?ID?NO：6)

AD3

CGFEELAWKIAKMIWSDVFQQGC(SEQ?ID?NO：7)

In other optional embodiments, other sequence variants of AD and/or DDD part can be used for DNL ^tMthe structure of complex.For example, there are only four kinds of variants of mankind PKA DDD sequence, corresponding to the DDD part of PKA RI α, RII α, RI β and RII β.RII α DDD sequence is the basis of above-disclosed DDD1 and DDD2.Four kinds of mankind PKA DDD sequences are below shown.DDD sequence represents the residue 1-44 of RII α, the residue 12-61 of the residue 1-44 of RII β, RI α and the residue 13-66 of RI β.(sequence of noticing DDD1 obtains from slight modification of mankind PKA RII α DDD part.)

PKA?RIα

SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEAK(SEQ?ID?NO：8)

PKA?RIβ

SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEENRQILA(SEQ?ID?NO：9)

PKA?RIIα

SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ(SEQ?ID?NO：10)

PKA?RIIβ

SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER(SEQ?ID?NO：11)

The structure-function relationship of AD and DDD domain is the theme of research.(referring to such as Bums-Hamuro etc., 2005, Protein Sci14:2982-92; Carr etc., 2001, J Biol Chem276:17332-38; Alto etc., 2003, Proc Natl Acad Sci USA100:4445-50; Hundsrucker etc., 2006, Biochem J396:297-306; Stokka etc., 2006, Biochem J400:493-99; Gold etc., 2006, Mol Cell24:383-95; Kinderman etc., 2006, Mol Cell24:397-408, its full content is separately incorporated herein by reference.)

For example, Kinderman etc. (2006, Mol Cell24:397-408) studied the crystal structure of AD-DDD binding interactions and infer mankind DDD sequence contain a plurality of dimer form or AKAP in conjunction with in important conservative amino acid residues, below following lining out in SEQ ID NO:1.(referring to Kinderman etc., Fig. 1 of 2006, is incorporated herein by reference.) those skilled in the art will recognize that, when the sequence variants of design DDD sequence, people can expect to avoid changing any residue with underscore, and can be to making conserved amino acid replacement for dimerization and AKAP in conjunction with not too important residue.

SH IQ IPPG LTE LLQG YT VE VLRQQPPD LVE FA VE YFTR LREARA(SEQ?ID?NO：1)

As below more discussed in detail, 20 kinds of common L-aminoacid have been characterized to conserved amino acid replacement separately.Therefore, according to the data of Kinderman (2006) and conserved amino acid, replace, the potential optional DDD sequence based on SEQ ID NO:1 is shown in Table 1.In the table 1 of design, only considered the aminoacid replacement of high conservative.For example, only with charged residue, replace the residue with identical charges, with the residue with similar size, replace the residue with little side chain, only use other hydroxyl substituted hydroxy side chains etc.Due to the unique effect of proline for aminoacid secondary structure, proline is not replaced by other residues.A limited number of these potential optional DDD partial sequences are shown in below in SEQ ID NO:12 to SEQ ID NO:31.Those skilled in the art will recognize that, can utilize standard technique, for example, with commercially available peptide synthesizer or well-known side-directed mutagenesis, build in DDD part classification the optional material of restricted number hardly.Also can easily determine that aminoacid replacement is for the effect of AD part combination by standard binding assay, such as disclosed in (2003, Proc Natl Acad Sci USA100:4445-50) such as Alto.

Conserved amino acid in table 1.DDD1 (SEQ ID NO:1) replaces.Common sequence is disclosed as SEQ ID NO:87.

THIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：12)

SKIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFFRLREARA(SEQ?ID?NO：13)

SRIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：14)

SHINIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：15)

SHIQIPPALTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：16)

SHIQIPPGLSELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：17)

SHIQIPPGLTDLLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：18)

SHIQIPPGLTELLNGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：19)

SHIQIPPGLTELLQAYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：20)

SHIQIPPGLTELLQGYSVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：21)

SHIQIPPGLTELLQGYTVDVLRQQPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：22)

SHIQIPPGLTELLQGYTVEVLKQQPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：23)

SHIQIPPGLTELLQGYTVEVLRNQPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：24)

SHIQIPPGLTELLQGYTVEVLRQNPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：25)

SHIQIPPGLTELLQGYTVEVLRQQPPELVEFAVEYFTRLREARA(SEQ?ID?NO：26)

SHIQIPPGLTELLQGYTVEVLRQQPPDLVDFAVEYFTRLREARA(SEQ?ID?NO：27)

SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFLVEYFTRLREARA(SEQ?ID?NO：28)

SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFIVEYFTRLREARA(SEQ?ID?NO：29)

SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFVVEYFTRLREARA(SEQ?ID?NO：30)

SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVDYFTRLREARA(SEQ?ID?NO：31)

Alto etc. (2003, Proc Natl Acad Sci USA100:4445-50) the AD sequence of various AKAP albumen is carried out to bioinformatic analysis and with design, be called as the RII selectivity AD sequence (SEQ ID NO:3) of AKAP-IS, wherein the binding constant of DDD is 0.4nM.AKAP-IS sequence is designed to be incorporated into the peptide antagonists of the AKAP of PKA.Below in SEQ ID NO:3, with underscore, marked the residue that tends to reduce the replacement of being combined with DDD in AKAP-IS sequence.Those skilled in the art will recognize that, when the sequence variants of design AD sequence, people can expect to avoid changing any residue with underscore, and can replace making conserved amino acid for DDD in conjunction with not too important residue.The potential conserved amino acid that table 2 shows in AKAP-IS sequence (AD1, SEQ ID NO:3) replaces, with in table 1 above about similar shown in DDD1 (SEQ ID NO:1).

Below SEQ ID NO:32 to SEQ ID NO:49 shows a limited number of these potential optional AD partial sequences.In addition the suitable material of big figure in the possible AD partial sequence classification of data manufacture, test and use that, those skilled in the art can be based on (2003) such as Alto.Notice, Fig. 2 of Alto (2003) shows can be based on actual in conjunction with testing manufacture and the maintenance simultaneously potential aminoacid replacement with the even very big figure of the combination activity of DDD part.

AKAP-IS

QIEYL AKQ IVDN AIQQA(SEQ?ID?NO：3)

Conserved amino acid in table 2.AD1 (SEQ ID NO:3) replaces.Common sequence is disclosed as SEQ ID NO:88.

NIEYLAKQIVDNAIQQA(SEQ?ID?NO：32)

QLEYLAKQIVDNAIQQA(SEQ?ID?NO：33)

QVEYLAKQIVDNAIQQA(SEQ?ID?NO：34)

QIDYLAKQIVDNAIQQA(SEQ?ID?NO：35)

QIEFLAKQIVDNAIQQA(SEQ?ID?NO：36)

QIETLAKQIVDNAIQQA(SEQ?ID?NO：37)

QIESLAKQIVDNAIQQA(SEQ?ID?NO：38)

QIEYIAKQIVDNAIQQA(SEQ?ID?NO：39)

QIEYVAKQIVDNAIQQA(SEQ?ID?NO：40)

QIEYLARQIVDNAIQQA(SEQ?ID?NO：41)

QIEYLAKNIVDNAIQQA(SEQ?ID?NO：42)

QIEYLAKQIVENAIQQA(SEQ?ID?NO：43)

QIEYLAKQIVDQAIQQA(SEQ?ID?NO：44)

QIEYLAKQIVDNAINQA(SEQ?ID?NO：45)

QIEYLAKQIVDNAIQNA(SEQ?ID?NO：46)

QIEYLAKQIVDNAIQQL(SEQ?ID?NO：47)

QIEYLAKQIVDNAIQQI(SEQ?ID?NO：48)

QIEYLAKQIVDNAIQQV(SEQ?ID?NO：49)

Gold etc. (2006, Mol Cell24:383-95) utilize crystallography and peptide screening to develop SuperAKAP-IS sequence (SEQ ID NO:50), represent the RII isoform of PKA than high five orders of magnitude of selectivity of RI isoform.Not underlined residue indication aminoacid replacement is with respect to the position of AKAP-IS sequence, and it has increased the combination with the DDD part of RII α.In this sequence, N end Q residue is numbered as residue numbering 4, and C end A residue is residue numbering 20.The residue that can replace to affect for the affinity of RII α is residue 8,11,15,16,18,19 and 20 (Gold etc., 2006).Expection, in some optional embodiment, can replace AKAP-IS AD partial sequence by SuperAKAP-IS sequence and prepare DNL ^tMconstruct.Other optional sequences that can replace AKAP-IS AD sequence are shown in SEQ ID NO:51-53.Replacement with respect to AKAP-IS sequence marks through underscore.Can expect, the same with the AD2 sequence shown in SEQ ID NO:4, AD partly also can comprise other N end residue cysteine and glycine and C end residue glycine and cysteine.

SuperAKAP-IS

QIEY VAKQIVD YAI HQA(SEQ?ID?NO：50)

Optional AKAP sequence

QIEY KAKQIVD HAI HQA(SEQ?ID?NO：51)

QIEY HAKQIVD HAI HQA(SEQ?ID?NO：52)

QIEY VAKQIVD HAI HQA(SEQ?ID?NO：53)

Fig. 2 of Gold etc. discloses other DDD binding sequences from multiple AKAP albumen, is shown in below.

rII specificity AKAP

AKAP-KL

PLEYQAGLLVQNAIQQAI(SEQ?ID?NO：54)

AKAP79

LLIETASSLVKNAIQLSI(SEQ?ID?NO：55)

AKAP-Lbc

LIEEAASRIVDAVIEQVK(SEQ?ID?NO：56)

rI-specificity AKAP

AKAPce

ALYQFADRFSELVISEAL(SEQ?ID?NO：57)

RIAD

LEQVANQLADQIIKEAT(SEQ?ID?NO：58)

PV38

FEELAWKIAKMIWSDVF(SEQ?ID?NO：59)

dual specificity AKAP

AKAP7

ELVRLSKRLVENAVLKAV(SEQ?ID?NO：60)

MAP2D

TAEEVSARIVQVVTAEAV(SEQ?ID?NO：61)

DAKAP1

QIKQAAFQLISQVILEAT(SEQ?ID?NO：62)

DAKAP2

LAWKIAKMIVSDVMQQ(SEQ?ID?NO：63)

Stokka etc. (2006, Biochem J400:493-99) have also developed the peptide competition thing of the AKAP that is incorporated into PKA, are shown in SEQ ID NO:64-66.Peptide antagonists is appointed as to Ht31 (SEQ ID NO:64), RIAD (SEQ ID NO:65) and PV-38 (SEQ ID NO:66).Ht-31 peptide represents for the affinity of the RII isoform of PKA larger, and RIAD and PV-38 demonstration are higher to the affinity of RI.

Ht31

DLIEEAASRIVDAVIEQVKAAGAY(SEQ?ID?NO：64)

RIAD

LEQYANQLADQIIKEATE(SEQ?ID?NO：65)

PV-38

FEELAWKLAKMIWSDVFQQC(SEQ?ID?NO：66)

Hundsrucker etc. (2006, Biochem J396:297-306) have developed other peptides competition things of the AKAP that is incorporated into PKA, for the binding constant of the DDD of the RII form of PKA, are low to moderate 0.4nM.The sequence of various AKAP antagonistic peptides is provided in, in the table 1 of Hundsrucker etc., to be below reproduced in table 3.AKAPIS represents synthetic RII subunit binding peptide.Shown in every other peptide is all derived from, the RII of AKAP is in conjunction with territory.

Table 3.AKAP peptide sequence

Below by underline to indicate the conservative residue of AD domain camber at different AKAP albumen for AKAP IS sequence (SEQ ID NO:3).Observed the same of described residue and Alto etc. (2003), has wherein added C end alanine residue.(referring to the Fig. 4 of (2006) such as Hundsrucker, be incorporated herein by reference.) peptide antagonists of special high-affinity of sequence have to(for) RII DDD sequence is the sequence of AKAP-IS, AKAP7 δ-wt-pep, AKAP7 δ-L304T-pep and AKAP7 δ-L308D-pep.

AKAP-IS

QIEYL AKQ IVDN AIQQ A(SEQ?ID?NO：3)

Carr etc. (2001, J Biol Chem276:17332-38) studied different AKAP from the mankind and non-human protein in conjunction with the sequence homology degree between DDD sequence, and identified the residue seeming in the conservative DDD sequence of topnotch in different DDD parts.These are below underlining to indicate by the mankind PKA RII α DDD sequence about SEQ ID NO:1.Conservative especially residue is further indicated by italic.It is overlapping but not identical that described residue and Kinderman etc. (2006) propose those residues important for being bonded to AKAP albumen.Those skilled in the art will recognize that, when the sequence variants of design DDD, most preferably avoid changing the most conservative residue (italic), and preferably avoid changing conserved residues (underlining), and can consider to carry out conserved amino acid replacement for the residue that does not both underline non-italic.

S HIQ IPP GLT ELLQGYTV EVLRQ QPP DLVEFAVE YFTR LR EA RA(SEQ?ID?NO：1)

One group of the DDD1 of the data based on (2001) such as Carr (SEQ ID NO:1) sequence replaces and is shown in Table 4 through the conserved amino acid of modifying.Even if that reduces by this group is substituted sequence, still exist 65,000 kinds above can be by those skilled in the art in the possible optional DDD partial sequence without producing, test and use under too much experiment.Those skilled in the art can easily obtain as disclosed these optional DDD aminoacid sequences of table 1 and table 2 above.

Conserved amino acid in table 4.DDD1 (SEQ ID NO:1) replaces.Common sequence is disclosed as SEQ ID NO:89.

Those skilled in the art will recognize that, use the standard technique in this area and only pass through normal experiment, these and other aminoacid replacement in DDD or AD aminoacid sequence can be used for being created in the optional material in AD or DDD part classification.

Interferon and other immunomodulators

In certain preferred aspects, effector is partly immunomodulator.Immunomodulator is a kind of immune dose of can change, suppress or stimulating health when existing.The immunomodulator using can comprise cytokine, stem cell factor, lymphotoxin, hematopoietic factor, colony stimulating factor (CSF), interferon (IFN), erythropoietin, thrombopoietin and its combination.Useful especially is lymphotoxin if tumor necrosis factor (TNF), hematopoietic factor are if interleukin (IL), colony stimulating factor are if granulocyte colony-stimulating factor (G-CSF) or granulocyte macrophage colony stimulating factor (GM-CSF), interferon are as interferon-' alpha ', interferon-beta, interferon-γ or interferon-λ, and stem cell factor is as being called " the S1 factor " person.

In a more preferred embodiment, effector is partly cytokine, for example lymphokine, monokine, somatomedin and traditional polypeptide hormone.Cytokine comprises that growth hormone is as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; Parathyroid hormone; Thyroxine; Insulin; Proinsulin; Relaxin; Relaxation precipitinogen; Glycoprotein hormones is as follicule-stimulating hormone (FSH) (FSH), thyrotropin (TSH) and interstitialcellstimulating hormone (ICSH) (LH); Placental growth factor (PIGF), liver growth factor; Prostaglandin, fibroblast growth factor; Lactotropin; Human placental lactogen, OB albumen; Tumor necrosis factor-alpha and tumor necrosis factor-β; Mullerian inhibiting substance; Mice promoting sexual gland hormone related peptides; Inhibin; Activin; VEGF; Integrin; Thrombopoietin (TPO); Nerve growth factor is as NGF-β; PDGF; Transforming growth factor (TGF) is as TGF-α and TGF-β; Insulin like growth factor-1 and insulin like growth factor-1 I; Erythropoietin (EPO); Bone-inducing factor; Interferon is as interferon-' alpha ', interferon-beta, interferon-λ and interferon-λ; Colony stimulating factor (CSF) is as macrophage-CSF (M-CSF); Interleukin (IL) for example, as IL-1, IL-1 α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit part or FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factor (TNF, TNF-α) and LT.In an especially preferred embodiment, cytokine is IFN-α 2b.

Protein or peptide immunomodulator, if the aminoacid sequence of cytokine is well-known in the art, and can be used any such known array in practice of the present invention.Those skilled in the art understand the multiple source of the public information of cytokine sequence.For example, the protein that ncbi database comprises a large amount of cytokines and immunomodulator and nucleic acid sequence encoding, for example erythropoietin (GenBank NM000799), IL-1 β (GenPept AAH08678), GM-CSF (GenPept AAA52578), TNF-α (GenPept CAA26669), interferon-' alpha ' (GenPept AAA52716.1), interferon-' alpha ' 2b (GenPept AAP20099.1), interferon-λ (GenPept30G6_B; 3HHC_A; 3HHC_B; 3HHC_C; 3HHC_D; EAW56870.1; EAW56869.1; AAI40873.1) and any in fact listed peptide or protein immunomodulator above.Identify any interested protein or the suitable aminoacid of peptide effector part and/or the customary affairs that nucleotide sequence is those skilled in the art substantially.The commercial source of cytokine also can obtain and can use, for example total length mankind IFN-α 2b cDNA clone (the human cloned catalogue #HORF01 clone of Invitrogen Ultimate ORF ID IOH35221).

Antibody

In certain embodiments, antibody or its Fab can be incorporated to DNL ^tMin construct, for example, by antibody or fragment being connected to interferon or other cytokines for cytokine targeted delivery.Any known antibody or its Fab can be incorporated to DNL ^tMin construct.In preferred embodiments, complex is used for to cancer therapy and antibodies in tumor associated antigen (TAA).Kinds of tumors related antigen is as known in the art, includes, but is not limited to carbonic anhydrase IX, CCCL19, CCCL21, CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, AFP, PSMA, CEACAM5, CEACAM6, B7, the ED-B of fibronectin, factor H, FHL-1, Flt-3, folacin receptor, GROB, HMGB-1, hypoxia inducible factor (HIF), HM1.24, insulin-like growth factor-i (ILGF-1), IFN-γ, IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10, MAGE, mCRP, MCP-1, MIP-1A, MIP-13, MIF, MUC1, MUC2, MUC3, MUC4, MUC5a, PAM4 antigen, NCA-95, NCA-90, Ia, HM1.24, EGP-1, EGP-2, HLA-DR, tenascin (tenascin), Le (y), RANTES, T101, TAC, Tn antigen, Thomson-Friedenreich antigen, neoplasm necrosis antigen, TNF-α, TRAIL receptor (R1 and R2), VEGFR, EGFR, PlGF, complement factor C3, C3a, C3b, C5a, C5 and oncogene products.The target antigen of other types can be used for the therapy based on antibody of various disease state and can utilize the DNL of the antibody that is associated with the optional antigen of any this class of targeting ^tMconstruct.

Can be used for DNL ^tMexemplary anticancrin in construct includes, but is not limited to hR1 (anti-IGF-1R, U.S. Provisional Patent Application sequence No.61/145 896, are filed in 1/20/09), hPAM4 (anti-MUC1, U.S. Patent No. 7,282,567), hA20 (anti-CD20, U.S. Patent No. 7,251,164), hA19 (anti-CD19, U.S. Patent No. 7,109,304), hIMMU-31 (anti-AFP, U.S. Patent No. 7,300,655), hLL1 (anti-CD74, U.S. Patent No. 7,312,318), hLL2 (anti-CD22, U.S. Patent No. 7,074,403), hMu-9 (anti-CSAp, U.S. Patent No. 7,387,773), hL243 (anti-HLA-DR, U.S. Patent No. 7,612,180), hMN-14 (anti-CEACAM5, U.S. Patent No. 6,676,924), hMN-15 (anti-CEACAM6, U.S. Patent application No.10/672,278), hRS7 (anti-EGP-1, U.S. Patent No. 7,238,785) and hMN-3 (anti-CEA, U.S. Patent No. 7,541,440), the embodiment of each institute's referenced patents or application is partly incorporated herein by reference.Those skilled in the art will recognize that, this list not tool is restricted and any other known anti-TAA antibody can be incorporated to DNL ^tMin construct.

Antigen binding antibody fragment is well known in the art, for example F (ab ') ₂, F (ab) ₂, Fab ', Fab, Fv, scFv etc., and can use any this type of known fragment.As used herein, antigen binding antibody fragment refers to that combination can be by any fragment of the antibody of the same antigen of complete or parental antibody identification.Become known for any in fact interested antibody of preparation or the AD of fragment and/or the technology of DDD conjugates (for example, U.S. Patent No. 7,527,787).

Spendable not yoke closes in the antibody of therapeutic agent or its fragment and is called as " naked " antibody or its fragment.In optional embodiment, antibody or fragment conjugate can be closed in one or more therapeutic agents and/or diagnostic agent.Be known in the art extensively multiple this type of therapeutic agent and diagnostic agent, as below more discussed in detail, and can use any this type of known therapeutic agent or diagnostic agent.

For the preparation of resisting the technology of the monoclonal antibody of any in fact target antigen, be well known in the art.Referring to for example Kohler and Milstein, Nature256:495 (1975); And Coligan etc. (volume), CURRENT PROTOCOLS IN IMMUNOLOGY, the 1st volume, 2.5.1-2.6.7 page (John Wiley & Sons 1991).Concise and to the point, can be by the following monoclonal antibody that obtains: the compositions that comprises antigen to injection in mice, shift out spleen to obtain bone-marrow-derived lymphocyte, bone-marrow-derived lymphocyte and myeloma cell are merged to generation hybridoma, clone described hybridoma, select to produce the positive colony for the antibody of antigen, cultivate described generation for the clone of the antibody of antigen, and described antibody is separated from hybridoma culture.

Can be by multiple perfect technology by MAb separation and purification from hybridoma culture.This type of isolation technics comprises affinity chromatography, size exclusion chromatography (SEC) and the ion exchange chromatography that uses protein A agarose.2.7.1-2.7.12 page and 2.9.1-2.9.3 page referring to for example Coligan.In addition, referring to Baines etc., " Purification ofImmunoglobulin G (IgG), " METHODS IN MOLECULAR BIOLOGY, the 10th volume, 79-104 page (The Humana Press, Inc.1992).

After initially producing for immunogenic antibody, then antibody order-checking can be prepared by recombinant technique.Humanization and chimericization of the well-known rodent antibody of those skilled in the art and antibody fragment.Use is derived from humanization, antibody ingredient chimeric or human antibodies has been got rid of the potential problems relevant to the immunogenicity of muroid constant region.

Chimeric antibody

Chimeric antibody is a kind of recombiant protein, and wherein the variable region of human antibodies is replaced by the variable region of for example mouse antibodies (complementary determining region (CDR) that comprises mouse antibodies).When being applied to experimenter, chimeric antibody shows immunogenicity reduction and stability increases.The general technology of clone's muroid immunoglobulin variable domain is disclosed in such as Orlandi etc., in Proc.Nat ' l Acad.Sci.USA86:3833 (1989).Those skilled in the art build the technology of chimeric antibody as everyone knows.For example, Leung etc., in Hybridoma13:469 (1994), by the V of the muroid LL2 that will encode (a kind of Anti-CD22 monoclonal antibody) _κand V _hthe DNA sequence of domain and corresponding mankind κ and IgG ₁constant region domain combination and produce LL2 chimera.

Humanized antibody

For generation of the technology of humanization MAb, be well known in the art (referring to such as Jones etc., Nature321:522 (1986); Riechmann etc., Nature332:323 (1988); Verhoeyen etc., Science239:1534 (1988); Carter etc., Proc.Nat ' l Acad.Sci.USA89:4285 (1992); Sandhu, Crit.Rev.Biotech.12:437 (1992); And Singer etc., J.Immun.150:2844 (1993)).Corresponding variable domains that can be by the mice CDR of the heavy variable chains of mouse immuning ball protein and light variable chains is transferred to human antibodies is by chimeric or muroid monoclonal antibody human source.Mice framework region (FR) in chimeric mAb is also replaced by mankind FR sequence.Owing to simply mice CDR being transferred in mankind FR and often causes the reduction of Antibody avidity even to be lost, therefore may need extra modification to recover the original affinity of rodent antibody.This can be replaced into by the one or more mankind's residues in JiangFR district its muroid homologue and obtain the antibody that its epi-position is had to a good combination affinity and realize.Referring to such as Tempest etc., Biotechnology9:266 (1991) and Verhoeyen etc., Science239:1534 (1988).Conventionally, mankind FR amino acid residues different from muroid homologue and that be positioned near to or in contact with one or more cdr amino acid residues will become the candidate of replacement.

Human antibodies

The method that the transgenic animal of using combined method or transforming with human immunoglobulin gene seat produce complete human antibodies is (for example, Mancini etc., 2004, New Microbiol.27:315-28 as known in the art; Conrad and Scheller, 2005, Comb.Chem.High Throughput Screen.8:117-26; Brekke and Loset, 2003, Curr.Opin.Phamacol.3:544-50).Complete human antibodies also can and be had a liking for thalline display technique by gene or chromosome transfection method and build, and described technology is all as known in the art.Referring to such as McCafferty etc., Nature348:552-553 (1990).This type of complete human antibodies expection represents than chimeric or humanized antibody side effect still less, and brings into play the function of endogenous human antibodies substantially in vivo.In certain embodiments, method required for protection and program can be utilized the human antibodies producing by this type of technology.

In another case, can use display technique of bacteriophage generate human antibodies (for example, Dantas-Barbosa etc., 2005, Genet.Mol.Res.4:126-40).Can or show particular disease states from normal human subject and generate human antibodies (Dantas-Barbosa etc., 2005) as the mankind of cancer.The advantage that builds human antibodies from ill individuality is that circulating antibody storehouse may have a preference for the antibody of anti-disease association antigen.

In a limiting examples of this method, Dantas-Barbosa etc. (2005) have built the phage display library from the mankind Fab antibody fragment of Patients with Osteosarcoma.Conventionally, total RNA obtains (the same) by blood circulation lymphocyte.From the antibody library of μ, γ and κ chain, clone recombinant Fab and insert (the same) phage display library.By RNA be converted into eDNA and for use for the Auele Specific Primer of heavy chain and light chain immunoglobulin sequences produce Fab cDNA library (Marks etc., 1991, J.Mol.Biol.222:581-97).According to Andris-Widhopf etc. (2000, in Phage Display Laboratory Manual, Barbas etc. (volume), the 1st edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, the 9.1st to 9.22 pages) carry out the structure in library.By restriction endonuclease, digest final Fab fragment and insert phage genome and generate phage display library.Can screen described library (referring to for example Pasqualini and Ruoslahti, 1996, Nature380:364-366 by standard phage display method as known in the art; Pasqualini, 1999, The Quart.J.Nuel.Med.43:159-162).

Phage display can carry out by multiple format, and it is summarized referring to for example Johnson and Chiswell, Current Opinion in Structural Biology3:5564-571 (1993).Also can pass through the B Hemapoiesis human antibodies of Activated in Vitro.Referring to U.S. Patent No. 5,567,610 and 5,229,275, by the mode quoting in full, be incorporated herein.Those skilled in the art will recognize that, these technology are exemplary, and can utilize any known to the method with screening human antibodies or antibody fragment.

In the optional situation of another kind, usable criterion immunization protocol, is used and to produce the transgenic animal of human antibodies, is generated the anti-antibody of any immunogenicity target substantially by genetically engineered.The method that obtains human antibodies from transgenic mice is disclosed in Green etc., Nature Genet.7:13 (1994); Lonberg etc., Nature368:856 (1994); And Taylor etc., in Int.Immun.6:579 (1994).A limiting examples of this kind of system is from Abgenix's (Fremont, CA) (for example, Green etc., 1999, J.Immunol.Methods231:11-23).? in similar animal, mouse antibodies gene has been inactivated and has substituted with functional human antibody gene, and the maintenance of the remainder of mouse immune system is complete.

The YAC (yeast artificial chromosome) of the system genitale structure of the part that use contains mankind IgH and Ig kappa gene seat (comprising most of variable region sequences and auxiliary gene and regulating and controlling sequence) transforms .Storehouse, mankind variable region can be used for generating the B cell that produces antibody, and it can be treated as hybridoma by known technology.Utilize target antigen immunity by normal immunoreaction, will produce human antibodies, it can be gathered and/or be prepared by standard technique discussed above.Multiple strain is available, and each can both produce different types of antibody.The human antibodies that transgenic produces has shown to have treatment potentiality, keeps the pharmacokinetic property (Green etc., 1999) of normal human subject antibody simultaneously.Those skilled in the art will recognize that, compositions required for protection and method are not limited to use system, and can utilize by genetically engineered any transgenic animal with generation human antibodies.

Antibody fragment

Can generate by known technology the antibody fragment of identification specificity epi-position.Antibody fragment is the antigen-binding portion thereof of antibody, for example F (ab ') ₂, Fab ', F (ab) ₂, Fab, Fv, sFv etc.F (ab ') ₂fragment can be produced by pepsin digested antibody molecule, and Fab ' fragment can be by reduction F (ab ') ₂the disulfide bond of fragment and generating.Alternatively, to having, required specific monoclonal Fab ' fragment is carried out rapidly and easy evaluation to allow can to build Fab ' expression library (Huse etc., 1989, Science, 246:1274-1281).F (ab) ₂fragment can be generated by papain digestion antibody, and Fab fragment can be obtained by Reduction of Disulfide.

ScFv molecule (scFv) comprises VL domain and VH domain.VL and VH domain associate and form target binding site.These two domains are further covalently bound by peptide linker (L).The method of the peptide linker that preparation scFv molecule and design are suitable is in following description: U.S. Patent No. 4,704,692; U.S. Patent No. 4,946,778; R.Raag and M.Whitlow, " Single Chain Fvs. " FASEB the 9th volume: 73-80 (1995); And R.E.Bird and B.W.Walker, " Single Chain Antibody Variable Regions, " TIBTECH, the 9th volume: 132-137 (1991).

Technology for generation of single domain antibody (DAB) is also known in the art, such as disclosed in (2006, the Prot Express Purif51:253-259) such as Cossins being incorporated herein by reference.

Can be by full length antibody Proteolytic enzyme or the DNA that expresses encoding antibody fragment in escherichia coli (E.coli) or another kind of host be carried out to Dispersal risk fragment.Can use pepsin or papain digestion full length antibody to obtain antibody fragment by conventional method.These methods have been described in the U.S. Patent No. 4,036,945 and 4,331,647 of Goldenberg for example and wherein in contained list of references.In addition, referring to Nisonoff etc., Arch Biochem.Biophys.89:230 (1960); Porter, Biochem.J.73:119 (1959); Edelman etc., METHODS IN ENZYMOLOGY, the 1st volume, the 422nd page (Academic Press1967); And 2.8.1-2.8.10 page and the 2.10.-2.10.4 page of Coligan.

Known antibodies

Can be from the commercially available antibody used in extensive multiple known source.For example, by American type culture collection (ATCC, Manassas, VA), can obtain Multiple Antibodies secreted hybridoma cell line.The preservation and/or disclose variable region sequences and can be used in method and composition required for protection in ATCC of a large amount of antibody for various diseases target (including but not limited to tumor associated antigen).Referring to for example U.S. Patent No. 7,312,318; 7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509; 7,049,060; 7,045,132; 7,041,803; 7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598; 6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018; 6,964,854; 6,962,981; 6,962,813; 6,956,107; 6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547; 6,921,645; 6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625; 6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580; 6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226; 6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778; 6,812,206; 6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681; 6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,155; 6,716,966; 6,709,653; 6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852; 6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279; 6,596,852; 6,592,868; 6,576,745; 6,572,856; 6,566,076; 6,562,618; 6,545,130; 6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915; 6,488,930; 6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356; 6,455,044; 6,455,040; 6,451,310; 6,444,206; 6,441,143; 6,432,404; 6,432,402; 6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350; 6,383,759; 6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244; 6,346,246; 6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393; 6,254,868; 6,187,287; 6,183,744; 6,129,914; 6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540; 5,814,440; 5,798,229; 5,789,554; 5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953; 5,525,338.These are only for exemplary and be known in the art extensively multiple other antibody and its hybridoma.Those skilled in the art will recognize that, can for the antibody of interested selected disease association target, obtain for almost antibody sequence or the antibody-secreting hybridoma of any disease association antigen by simple search in ATCC, NCBI and/or USPTO data base.Use standard technique well known in the art, the antigen binding structural domain amplification of clonal antibody can be sheared, connect and enter expression vector, transfection enters and adapts to host cell and for generation of protein.

The specific antibodies that can be used for the cancer therapy in the scope of method and composition required for protection includes, but is not limited to LL1 (anti-CD74), epratuzumab (LL2) and RFB4 (anti-CD22), RS7 (anti-Glycoproteins in Epithelial-1 (EGP-1) or anti-TROP-2), PAM4 and KC4 (being all anti-stick albumen), MN-14 (anticancer embryonal antigen (CEACAM5, also referred to as CD66e), Mu-9 (anti-colon-specific antigen-p), Immu-31 (Anti-α-Fetoprotein), anti-TAG-72 (for example CC49), anti-Tn, J591 or HuJ591 (anti-PSMA (prostate specific membrane antigen)), AB-PG1-XG1-026 (anti-PSMA dimer), D2/B (anti-PSMA), G250 (anti-carbonic anhydrase IX), hL243 (anti-HLA-DR), alemtuzumab (anti-CD52), bevacizumab (anti-VEGF), Cetuximab (anti-EGFR), lucky trastuzumab (anti-CD 33), ibritumomab tiuxetan (anti-CD20), Victibix (anti-EGFR), Rituximab (anti-CD20), tositumomab (anti-CD20), GA101 (anti-CD20), dimension trastuzumab (anti-CD20), and Herceptin (anti-ErbB).These antibody are (for example, U.S. Patent No. 5,686,072,5,874,540,6,107,090,6 as known in the art, 183,744,6,306,393,6,653,104,6,730.300,6,899,864,6,926,893,6,962,702,7,074,403,7,230,084,7,238,785,7,238,786,7,256,004,7,282,567,7,300,655,7,312,318,7,585,491,7,612,180,7,642,239; With U.S. Patent Application Publication No.20040202666 (now waiving the right), 20050271671 and 20060193865; The embodiment of each document is partly incorporated herein by reference).The concrete known antibodies of using comprises hPAM4 (U.S. Patent No. 7, 282, 567), hA20 (U.S. Patent No. 7, 251, 164), hA19 (U.S. Patent No. 7, 109, 304), hIMMU31 (U.S. Patent No. 7, 300, 655), hLL1 (U.S. Patent No. 7, 312, 318), hLL2 (U.S. Patent No. 7, 074, 403), hMu-9 (U.S. Patent No. 7, 387, 773), hL243 (U.S. Patent No. 7, 612, 180), hMN-14 (U.S. Patent No. 6, 676, 924), hMN-15 (U.S. Patent No. 7, 541, 440), hR1 (U.S. Patent application 12/772, 645), hRS7 (U.S. Patent No. 7, 238, 785), hMN-3 (U.S. Patent No. 7, 541, 440), AB-PG1-XG1-026 (U.S. Patent application 11/983, 372, with ATCC PTA-4405 and PTA-4406, deposit) and D2/B (WO2009/130575), described in each, the full text of patent or application is incorporated herein in the mode of partly quoting about drawings and Examples.

Anti-TNF-α antibody is as known in the art and can be used for treating immunological diseases, for example asthma.The known antibody for TNF-α comprise human antibodies CDP571 (Ofei etc., 2011, Diabetes45:881-85); Rodent antibody MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI, M302B and M303 (Thermo Scientific, Rockford, IL); Infliximab (Centocor, Malvern, PA); Pegylation match trastuzumab (UCB, Brussels, Belgium); And adalimumab (Abbott, Abbott Park, IL).These and many other known anti-TNF-α antibodies can be used in method and composition required for protection.Other antibody that use include, but is not limited to anti-B cell antibody, for example, tie up trastuzumab, epratuzumab, meter La Zhu monoclonal antibody or hL243; Holder pearl monoclonal antibody (anti-IL-6 receptor); Basiliximab (anti-CD25); Daclizumab (anti-CD25); Pearl monoclonal antibody (anti-CD11a) in accordance with the law; Muromonab-CD3 (anti-CD3 receptor); Anti-CD 40 L (UCB, Brussels, Belgium); Natalizumab (anti-α 4 integrins) and omalizumab (anti-IgE).

Macrophage migration inhibitory factor (MIF) is congenital and adaptive immunity and apoptotic important regulatory factor.Reported that CD74 is the endogenous recipient (Leng etc., 2003, J Exp Med197:1467-76) of MIF.The anti-CD74 antibody of Antagonism can be used for treating extensive various disease states for the therapeutical effect of approach in the cell of MIF mediation, for example bladder cancer, carcinoma of prostate, breast carcinoma, pulmonary carcinoma, colon cancer and chronic lymphocytic leukemia are (for example, Meyer-Siegler etc., 2004, BMC Cancer12:34; Shachar and Haran, 2011, Leuk Lymphoma52:1446-54); Nephropathy, for example kidney allograft rejection reaction (Lan, 2008, Nephron Exp Nephrol.109:e79-83); And numerous inflammatory diseases (electronic publishing was on March 22nd, 2009 for Meyer-Siegler etc., 2009, Mediators Inflamm; Takahashi etc., 2009, Respir Res10:33).Meter La Zhu monoclonal antibody (hLL1) is the exemplary anti-CD74 antibody with the therapeutic use that is used for the treatment of Jie's MIF mediated diseases.

Pharmaceutical composition of the present invention can be used for treatment and has neurodegenerative disease as the experimenter of Alzheimer.A bar pearl monoclonal antibody is in the clinical trial for the treatment of of alzheimer's disease.Other antibody that are proposed to be used in treatment of alzheimer's disease comprise Alz50 (Ksiezak-Reding etc., 1987, J Biol Chem263:7943-47), more spit of fland reed monoclonal antibody and Su Lan pearl monoclonal antibody.Infliximab (a kind of anti-TNF-α antibody) has been in the news and can have reduced amyloid speckle and improve cognitive.Because CD74 expresses (Bryan etc., Mol Neurodegeneration2008 in the neurofibrillary tangles of Alzheimer brain in patients; 3:13), thus CD74 be anti-CD74 antagonistic peptide by independent use or antibody or make for treating the another kind of target of this disease with conjugates form by DNL construct as described herein.

Spendable other antibody comprise for infectious disease pathogen as the antibody of antibacterial, virus, mycoplasma or other pathogen.The much antibody for these infectious pathogens is that as known in the art and any this known antibody all can be used in method and composition required for protection.For example, for the antibody of the gp120 glycoprotein antigen of HIV (human immunodeficiency virus) I (HIV-1), be known, and some this antibody can have immanoprotection action in the mankind.Referring to such as Rossi etc., Proc.Natl.Acad.Sci.USA.86:8055-8058,1990.Known anti-HIV antibody comprises (AIDS.2006 October 3 by Johansson etc.; 20 (15): the anti-peplos antibody of 1911-5) describing, and the anti-HIV antibody of describing and selling by Polymun (Vienna, Austria), it is also described in United States Patent (USP) 5,831,034, United States Patent (USP) 5,911,989, and Vcelar etc., AIDS2007; 21 (16): 2161-2170 and Joos etc., Antimicrob.Agents Chemother.2006; 50 (5): in 1773-9, be all incorporated herein by reference.

Antibody for malarial parasite can be for zygoblast, merozoite, schizont and gametocyte stage.Generated the monoclonal antibody for zygoblast (Circumsporozoite antigen), and shown its in vitro with rodent in can in and zygoblast (N.Yoshida etc., Science207:71-73,1980).Some team have developed toxoplasma antibody, and toxoplasma is protozoon parasite (Kasper etc., J.Immunol.129:1694-1699,1982 related in toxoplasmosis; The same, 30:2407-2412,1983).Developed for the antibody of virgin worm surface antigen and found it in vivo or interaction in vitro in virgin worm (Simpson etc., Parasitology, 83:163-177,1981; Smith etc., Parasitology, 84:83-91,1982; Gryzch etc., J.Immunol., 129:2739-2743,1982:Zodda etc., J.Immunol.129:2326-2328,1982; Dissous etc., J.immunol., 129:2232-2234,1982).

Schizotrypanum cruzi is the pathogen of American trypanosomiasis, and hunts stinkbug entomochory by what suck blood.Generate specificity in vitro and suppressed parasitic a kind of form to the differentiation of another kind of form (epimastigote is to the trypomastigote stage) and the antibody reacting with cell surface glycoprotein; Yet there is not this antigen (Sher etc., Nature, 300:639-640,1982) in parasitic mammal (blood flow) form.

Anti fungal antibody is as known in the art, for example anti-sclerotiniose antibody (United States Patent (USP) 7,910,702); Anti-glucuronoxylose mannan antibody (Zhong and Priofski, 1998, Clin Diag Lab Immunol5:58-64); Anti-candida belong to antibody (Matthews and Burnie, 2001,2:472-76); And anti-glycosyl sphingolipid antibody (Toledo etc., 2010, BMC Microbiol10:47).

Developed the suitable antibodies that causes most of microorganism (antibacterial, virus, protozoacide, fungus, other parasites) of infecting for great majority in the mankind, and many previously for in-vitro diagnosis object.These antibody that can generate by conventional method and more novel antibodies are applicable in the present invention.

Antibody is special-shaped

The immunogenicity of therapeutic antibodies increases to infusion reaction risk and the therapeutic response persistent period reduces relevant (Baert etc., 2003, N Engl J Med348:602-08).Can partly measure therapeutic antibodies by the abnormal shape of antibody and in host, bring out immunoreactive degree (Stickler etc., 2011, Genes and Immunity12:213-21).In the special-shaped constant region sequence with antibody of antibody, the aminoacid sequence variation of ad-hoc location is relevant.The abnormal shape of the IgG antibody that comprises heavy chain γ type constant region is called Gm abnormal shape (1976, J Immunol117:1056-59).

For common IgG1 human antibodies, the most general abnormal shape is G1m1 (Stickler etc., 2011, Genes and Immunity12:213-21).Yet, in white people, also frequently there is G1m3 special-shaped (the same).Reported that G1m1 antibody contains special-shaped sequence, when being applied to non-G1m1 (nG1m1) acceptor as G1m3 patient, described special-shaped sequence tends to bring out immunoreation (the same).When being applied to G1m1 patient, non-G1m1 haterotypic antibody is not immunogenicity (the same).

Mankind G1m1 abnormal shape is included in the aminoacid aspartic acid of Kabat position 356 and at the leucine of Kabat position 358 in the CH3 of heavy chain IgG1 sequence.NG1m1 abnormal shape is included in the aminoacid glutamic acid of Kabat position 356 and at the methionine of Kabat position 358.G1m1 and nG1m1 abnormal shape are included in the glutaminic acid residue of Kabat position 357 and described abnormal shape is called as DEL sometimes and EEM is special-shaped.The limiting examples that has shown exemplary antibodies Rituximab (SEQ ID NO:85) and the G1m1 of dimension trastuzumab (SEQ ID NO:86) and the CH sequence of nG1m1 haterotypic antibody.

Rituximab weight chain variabl area sequence (SEQ ID NO:85)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGG

PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY

NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK

SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Dimension trastuzumab variable region of heavy chain (SEQ ID NO:86

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG

PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY

NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK

SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(2009, the characteristic sequence of mAb1:1-7) having commented IgG abnormal shape changes with it for immunogenic impact for Jefferis and Lefranc.They have reported that G1m3 abnormal shape is characterised in that the arginine residues of Kabat position 214, form contrast with the lysine residue of Kabat214 in G1m17 abnormal shape.NG1m1,2 abnormal shapes are characterised in that the alanine of the glutamic acid of Kabat position 356, the methionine of Kabat position 358 and Kabat position 431.G1m1,2 abnormal shapes are characterised in that the glycine of the aspartic acid of Kabat position 356, the leucine of Kabat position 358 and Kabat position 431.Except CH sequence variants, Jefferis and Lefranc (2009) have also reported the special-shaped variant of κ constant region of light chain, wherein Km1 abnormal shape is characterised in that the valine of Kabat position 153 and the leucine of Kabat position 191, Km1,2 abnormal shapes are characterised in that the alanine of Kabat position 153 and the leucine of Kabat position 191, and Km3 abnormal shape is characterised in that the alanine of Kabat position 153 and the valine of Kabat position 191.

About therapeutic antibodies, dimension trastuzumab and Rituximab are respectively by humanization and be used to the treatment of extensive multiple hematology malignant tumor for the chimeric IgG1 antibody of CD20.Table 5 has compared the special-shaped sequence of Rituximab with dimension trastuzumab.As shown in table 5, Rituximab (G1m17,1) is the special-shaped IgG1 of DEL, and wherein in Kabat position 214, the other sequence variation of (heavy chain CH1) be lysine in Rituximab, with respect to being arginine in tieing up trastuzumab.Reported the immunogenicity of dimension trastuzumab in experimenter lower than Rituximab (referring to such as Morchhauser etc., 2009, J Clin Oncol27:3346-53; Goldenberg etc., 2009, Blood113:1062-70; Robak and Robak, 2011, BioDrugs25:13-25), this effect is by the difference owing between humanized antibody and chimeric antibody.Yet the special-shaped difference between EEM and DEL abnormal shape also may cause the immunogenicity of dimension trastuzumab lower.

The abnormal shape of table 5. Rituximab to dimension trastuzumab

In order to reduce the immunogenicity of therapeutic antibodies in nG1m1 genotype individuality, wish to select corresponding to following antibody special-shaped: G1m3 is special-shaped, be characterised in that the arginine of Kabat214; And nG1m1,2 is empty special-shaped, is characterised in that the alanine of the glutamic acid of Kabat position 356, the methionine of Kabat position 358 and Kabat position 431.Surprisingly, find that the repetition subcutaneous administration of G1m3 antibody within a very long time do not produce significant immunoreation.In optional embodiment, there is the arginine of Kabat214, the methionine of the glutamic acid of Kabat356, Kabat359 and the alanine of Kabat431 with special-shaped the same IgG 4 heavy chains of G1m3.Because immunogenicity seems relevant with the residue of those positions at least to a certain extent, so be also a preferred embodiment by IgG 4 being used for the treatment of property of CH sequence antibody.The combination of G1m3IgG1 antibody and IgG4 antibody also can be used for therapeutic administration.

Aminoacid replacement

In certain embodiments, disclosed method and composition may relate to the protein of the amino acid residue with one or more replacements or the generation of peptide and use.In a limiting examples, for the manufacture of DNL ^tMthe DDD of construct and/or AD sequence can be further optimized, for example, to increase DDD-AD binding affinity.

It will be recognized by those skilled in the art, generally, aminoacid replacement typically relates to the another kind of amino acid replacement (that is, conserved amino acid replaces) with relative similar characteristics for a seed amino acid.Various amino acid whose characteristics and aminoacid replacement are furtherd investigate and are known in the art the impact of protein structure and function.

For example, hydrophilic index (Kyte and Doolittle, 1982, J.Mol.Biol., 157:105-132) that can considered amino acid.The secondary structure that amino acid whose relatively hydrophilic feature is facilitated gained protein, it defines again the interaction of described protein and other molecules conversely.Based on amino acid whose hydrophobicity and charge characteristic, to every seed amino acid, give a hydrophilic index (Kyte and Doolittle, 1982), be specially: isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine/cystine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (0.4); Threonine (0.7); Serine (0.8); Tryptophan (0.9); Tyrosine (1.3); Proline (1.6); Histidine (3.2); Glutamic acid (3.5); Glutamine (3.5); Aspartic acid (3.5); Agedoite (3.5); Lysine (3.9); And arginine (4.5).When carrying out conservative replacement, preferably use the aminoacid of hydrophilic index in ± 2, be more preferably in ± 1, and be even more preferably in ± 0.5.

Aminoacid replacement also can be considered the hydrophilic (for example, U.S. Patent No. 4,554,101) of amino acid residue.Amino acid residue is endowed hydrophilicity value: arginine (+3.0); Lysine (+3.0); Aspartic acid (+3.0); Glutamic acid (+3.0); Serine (+0.3); Agedoite (+0.2); Glutamine (+0.2); Glycine (0); Threonine (0.4); Proline (0.5.+-.1); Alanine (0.5); Histidine (0.5); Cysteine (1.0); Methionine (1.3); Valine (1.5); Leucine (1.8); Isoleucine (1.8); Tyrosine (2.3); Phenylalanine (2.5); Tryptophan (3.4).Preferably with thering are similar hydrophilic other aminoacid, carry out amino acid replacement.

Other consideration comprises the size of amino acid side chain.For example, conventionally preferably the aminoacid with tight side chain is not replaced as to aminoacid with huge side chain as tryptophan or tyrosine as glycine or serine.Various amino acid residues are also a kind of considerations on the impact of secondary protein structure.Pass through empirical research, different amino acid residues for protein domain take alpha-helix, beta sheet or the tendentious impact of reversion secondary structure determined and known in the art (referring to for example Chou and Fasman, 1974, Biochemistry, 13:222-245; 1978, Ann.Rev.Biochem., 47:251-276; 1979, Biophys.J., 26:367-384).

Based on described consideration and deep empirical research, built conserved amino acid replacement table and known in the art.For example: arginine and lysine; Glutamic acid and aspartic acid; Serine and threonine; Glutamine and agedoite; And valine, leucine and isoleucine.Alternatively: Ala (A) leu, ile, Val; Arg (R) gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys (C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H) asn, gln, lys, arg; Ile (I) Val, met, ala, phe, leu; Leu (L) Val, met, ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F) leu, Val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W) phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations about aminoacid replacement comprise whether this residue is positioned at protein interior or is exposed to solvent.For inner residue, conservative replacement comprises: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala and Gly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr; Tyr and Trp.(referring to the PROWL website of for example rockefeller.edu).For the residue that is exposed to solvent, conservative replacement comprises: Asp and Asn; Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala and Gly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu; Leu and Ile; Ile and Val; Phe and Tyr.(the same) built various substrate and carried out assisted Selection aminoacid replacement, for example PAM250 scoring substrate, Dayhoff substrate, Grantham substrate, McLachlan substrate, Doolittle substrate, Henikoff substrate, Miyata substrate, Fitch substrate, Jones substrate, Rao substrate, Levin substrate and Risler substrate (the same).

When determining aminoacid replacement, people also can consider the existence of intermolecular or intramolecular bond, for example for example, for example, between positively charged residue (His, Arg, Lys) and electronegative residue (Asp, the Glu) formation of ionic bond (salt bridge), or the disulfide bond between close cysteine residues.

As everyone knows in coded protein sequence by any other amino acid whose method of any aminoacid replacement, and be those skilled in the art's normal experiment affairs, for example, by side-directed mutagenesis or by oligonucleotide montage synthetic and that assembling coded amino acid replaces, enter expression vector establishment body.

Fit

In certain embodiments, the targeting moiety of use can be fit.Structure well known in the art and measure the method for fit binding characteristic.For example, this type of technical description is in U.S. Patent No. 5,582, and in 981,5,595,877 and 5,637,459, its embodiment separately is partly incorporated herein by reference.The fit method that preparation and screening are incorporated into interested particular target is well-known, and for example U.S. Patent No. 5,475,096 and U.S. Patent No. 5,270,163, and its embodiment separately is partly incorporated herein by reference.

Fit by any known method (comprising synthetic, restructuring and purification process) preparation, and can be used alone or with other, specific ligand combination of identical target tool be used.In general, need minimum approximately 3 nucleotide, preferably at least 5 nucleotide are realized specific binding.Sequence fit that is shorter than 10 bases can be feasible, but there are 10,20,30 or 40 nucleotide fit can be preferred.

Fit can synthesizing through separation, order-checking and/or amplification or with conventional DNA or RNA molecular forms.Alternatively, the interested fit oligomer that comprises modification.Conventionally any hydroxyl existing in fit can phosphonate ester base, phosphate-based displacement, is protected, or is activated to prepare being connected of other and other nucleotide, or can be bonded to solid support thing by yoke by the protecting group of standard.One or more phosphodiester bonds can be replaced by optional linking group, and for example P (O) O is by P (O) S, P (O) NR ₂, P (O) R, P (O) OR ', CO or CNR ₂displacement, wherein R is that H or alkyl (1-20C) and R ' they are alkyl (1-20C); In addition, this group can be connected to adjacent nucleotide by O or S.All connections in oligomer are without identical.

Affine body and Fynomer

Some optional embodiment can utilize affine body to replace antibody.Affine body is commercially available from Affibody AB (Solna, Sweden).Affine body is to serve as antibody analog and for the small protein matter in conjunction with target molecules.By carry out integration engineering on α coilin matter skeleton, produce affine body (Nord etc., 1995, Protein Eng8:601-8; Nord etc., 1997, Nat Biotechnol15:772-77).The design of affine body is that IgG based on comprising a-protein is in conjunction with triple helical binding structure (Nord etc., 1995 in territory; 1997).Can produce affine body (Nord etc., 1995 with multiple binding affinity by the randomization of the 13 related seed amino acids in conjunction with activity of the Fc at bacterioprotein A; 1997).After randomization, pcr amplification library clone is screened for the phage display by mutain in phagicin carrier.Can use phage display triage techniques (for example, Pasqualini and Ruoslahti, 1996, the Nature380:364-366 of standard; Pasqualini, 1999, Quart.J.Nucl.Med.43:159-162) for any known antigen, screen phage display library, to identify that one or more are for the affine body of target antigen.

Specific to HER2/neu tool ¹⁷⁷the affine body of Lu labelling has confirmed the xenograft (Tolmachev etc., 2007, Cancer Res67:2773-82) of targeted expression HER2 in body.Although the nephrotoxicity causing due to gathering of low-molecular-weight radio-labelled compound is a problem at first, but reduced kidney with albuminous Reversible binding, gather, be achieved the therapy based on radionuclide (the same) of carrying out with the affine body being labeled.

Recently having proved uses radiolabeled affine body for the feasibility (Tolmachev etc., 2011, Bioconjugate Chem22:894-902) of in-vivo tumour imaging.The NOTA yoke of maleimide derivative closes in the affine body of anti-HER2 and uses ¹¹¹in radioactive label (the same).Be applied to the mice of expressing the DU-145 xenograft of HER2, then carry out γ camera imaging, allow visual (the same) of xenograft.

Fynomer also can be incorporated into antagonist and have similar affinity and specific target antigen.Fynomer is the mankind Fyn SH3 domain of the skeleton based on as binding molecule assembling.Described Fyn SH3 domain is 63 the amino acid whose complete human proteins that have that can produce with high yield in antibacterial.The polyspecific that Fynomer can link together to obtain two or more different antigen targets to have affinity is in conjunction with albumen.Fynomer is commercially available available from COVAGEN AG (Zurich, Switzerland).

Those skilled in the art will recognize that, in the practice of method and composition required for protection, affine body or fynomer can be used as to targeted molecular.

Bispecific and multi-specificity antibody

Bi-specific antibody can be used in multiple biomedical applications.For example, have and can guide specific tumor cell by T cytolysis to the bi-specific antibody of the binding site of TCSA and T cell surface receptor.The bi-specific antibody of the CD3 epi-position on identification glioma and T cell has been successfully used to treat the cerebral tumor (Nitta etc., the Lancet.1990 in human patients; 355:368-371).The technology of closing for therapeutic agent yoke disclosed herein in certain embodiments, can be used as targeting moiety with compositions together with bispecific or multi-specificity antibody.

Numerous methods for generation of bispecific or multi-specificity antibody are known, and as disclosed in U.S. Patent No. 7,405,320 for example, the embodiment of described patent is partly incorporated herein by reference.Can produce bi-specific antibody by limbs hybridoma method, described method relates to the fusion of two kinds of different hybridomas, and described hybridoma produces monoclonal antibody (Milstein and Cuello, Nature, 1983 of the different antigen site of identification separately; 305:537-540).

Another kind of method for generation of bi-specific antibody is come chemical tie two kinds of different monoclonal antibodies (Staerz etc., Nature.1985 with isodigeranyl functional cross-link agent; 314:628-631; Perez etc., Nature.1985; 316:354-356).Can also be by two parent's monoclonal antibodies be reduced into corresponding half molecule separately, then mix and make it reoxidize to obtain hybrid structure producing bi-specific antibody (Staerz and Bevan.Proc Natl Acad Sci U S are A.1986; 83:1453-1457).Alternative dispensing means relates to uses suitable joint by the Fab ' fragment chemical crosslinking of two or three independent purification.(referring to for example european patent application 0453082).

Additive method comprises by different selected markers being transferred to corresponding parent's hybridoma via the derivative shuttle vector of retrovirus, merged subsequently (DeMonte etc., Proc Natl Acad Sci U S A.1990,87:2941-2945); Or with the expression plasmid transfection hybridoma cell line of the heavy chain that comprises different antibodies and light chain gene, improve the efficiency that produces heterozygosis hybridoma.

The available peptide linker (being conventionally comprised of 12 above amino acid residues) with suitable composition and length is by homology V _hand V _ldomain is bonded together to form to be had in conjunction with active scFv (scFv).The method of manufacturing scFv is disclosed in U.S. Patent No. 4,946,778 and U.S. Patent No. 5,132,405 in, its embodiment separately is partly incorporated herein by reference.Peptide linker contraction in length can be prevented to the V on same chain to being less than 12 amino acid residues _hand V _ldomain matches and forces the V on other chains with complementary structure territory _hand V _ldomain pairing, thus cause forming functional polymer.The V engaging with the joint with 3 to 12 amino acid residues _hand V _lthe polypeptide chain of domain mainly forms dimer (being called double-chain antibody).Utilization has the joint of 0 to 2 amino acid residue, be conducive to form trimer (being called three chain antibodies) and the tetramer (being called four chain antibodies), but except joint length, definite oligomerization pattern seems to depend on composition and the orientation (V of V domain _h-joint-V _lor V _l-joint-V _h).

These technology for generation of polyspecific or bi-specific antibody are representing all difficulties aspect the labor-intensive of low-yield, purification necessity, low stability or technology.Recently, utilized and be called as combination that the technology of " stopping and locking (DNL) " produces any in fact required antibody, antibody fragment and other effector molecules (referring to for example U.S. Patent No. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and USSN11/925,408, its embodiment separately is partly incorporated herein by reference).Described technology utilization is called as anchoring structure territory (AD) and dimerization and stops the complementary protein binding domain of domain (DDD), they are bonded to each other and realize the assembling of composite structure, and formation scope comprises dimer, trimer, the tetramer, pentamer and six aggressiveness.These form stable complex with high yield and without extensive purification.DNL technology allows the assembling of monospecific, bispecific or multi-specificity antibody.Any technology that becomes known for manufacturing bispecific or multi-specificity antibody in this area can be used in the practice of the present invention's method required for protection.

Pre-targeting

Bispecific or multi-specificity antibody can be used in pre-targeting technology.Pre-targeting is the multistage method that initial exploitation is removed with the slow blood that solves direct targeting antibodies, and its normal tissue is less desirable toxicity as bone marrow causes.By pre-targeting, radionuclide or other treatment agent are connected to removed from blood in several minutes little and send molecule (can targeting construct).First use to can targeting construct and target antigen there is pre-targeting bispecific or the multi-specificity antibody of binding site, free antibody is removed from circulation, then using can targeting construct.

Pre-targeted approach is disclosed in such as people such as Goodwin, U.S. Patent No. 4,863,713; The people such as Goodwin, J.Nucl.Med.29:226,1988; The people such as Hnatowich, J.Nuch Med.28:1294,1987; The people such as Oehr, J.Nucl.Med.29:728,1988; The people such as Klibanov, J.Nucl.Med.29:1951,1988; The people such as Sinitsyn, J.Nucl.Med.30:66,1989; The people such as Kalofonos, J.Nucl.Med.31:1791,1990; The people such as Schechter, Int.J.Cancer48:167,1991; The people such as Paganelli, Cancer Res.51:5960,1991; The people such as Paganelli, Nucl.Med.Commun.12:211,1991; U.S. Patent No. 5,256,395; The people such as Stickney, Cancer Res.51:6650,1991; The people such as Yuan, Cancer Res.51:3119,1991; U.S. Patent No. 6,077,499; 7,011,812; 7,300,644; 7,074,405; 6,962,702; 7,387,772; 7,052,872; 7,138,103; 6,090,381; 6,472,511; 6,962,702; With 6,962,702, be incorporated herein by reference separately.

Can be by disease in experimenter for the treatment of or diagnosis or the pre-targeted approach of disease be provided below: (1) uses bi-specific antibody or antibody fragment to experimenter; (2) optionally to described experimenter, use removing compositions, and make described compositions remove antibody from circulation; And (3) to described experimenter use comprise one or more chelatings or chemically combined therapeutic agent or diagnostic agent as interferon lambda can targeting construct.

Can targeting construct

In certain embodiments, can select by one or more therapeutic agents or diagnostic agent labelling for pre-targeting can targeting construct peptide to be incorporated into thering are one or more binding sites and the target antigen relevant to disease or condition of illness be there is to the bi-specific antibody of one or more binding sites by targeting construct peptide.Bi-specific antibody can be used in pre-targeting technology, wherein can be first to experimenter's administration of antibodies.Enough time can be allowed so that bi-specific antibody is incorporated into target antigen and unconjugated antibody is removed from circulation.Then can to experimenter use can targeting construct as the peptide being labeled and make it be incorporated into bi-specific antibody and be positioned at diseased cells or tissue.

This type of can targeting construct can have various structures and not only can the antibody of targeting construct or the availability of fragment for being incorporated into high-affinity, and for when the use in pre-targeted approach and bi-specific antibody (bsAb) or multi-specificity antibody fast in body clearance rate select.Water-repelling agent is good at and causes strong immunoreation most, and hydrophilizing agent is preferred for removing in quick body.Therefore, equilibrium establishment between hydrophobic characteristics and hydrophilic characteristics.This can partly realize by offset the intrinsic hydrophobicity of many organic moiety with hydrophilic chelating agen.In addition, can select to have contrary dissolution properties can targeting construct subunit, peptide for example, it contains aminoacid, some of them tool hydrophobicity and some of them tool hydrophilic.

Can use to have less to two amino acid residues, the preferred peptide of 2 to 10 residues, and it also can be coupled to other parts as chelating agen.Joint should be low-molecular-weight conjugates, preferably has and is less than 50,000 dalton and is advantageously less than approximately 20,000 dalton, 10,000 dalton or 5,000 daltonian molecular weight.Can more generally will there is four or more residue by targeting construct peptide, for example PEPD OTA-Phe-Lys (HSG)-Tyr-Lys (HSG)-NH ₂(SEQ ID NO:98), wherein DOTA is Isosorbide-5-Nitrae, 7,10-tetraazacyclododecanand Isosorbide-5-Nitrae, 7,10-tetraacethyl and HSG are histamine succinyl glycyl.Alternatively, DOTA can be by NOTA (1,4,7-tri-azepines-cyclononane-1,4,7-triacetic acid), TETA (to acetyl bromide amido-benzyl-triethylammonium tetrakis tetraacethyl), NETA ([2-(the two carboxyl methyl [1 of 4,7-, 4,7] 7-triazacyclononane-1-base-ethyl]-2-carbonyl methyl-amino] acetic acid) or other known chelating moieties replacements.Can use chelating moiety for example to be incorporated into therapeutic and or diagnostic radionuclide, paramagnetic ion or contrast agent.

Can also can in backbone structure, comprise alpha-non-natural amino acid if D-aminoacid is to increase peptide stability in vivo by targeting construct.In optional embodiment, can use other backbone structures, those backbone structures that for example built by alpha-non-natural amino acid or class peptide.

Should use solid phase support thing and repeat quadrature deprotection and the standard technique of coupling on automated peptide synthesizer synthetic be used as can targeting construct peptide.By in peptide, be used to after a while free amine group that yoke closes chelating moiety or other agent advantageously by standard protecting group as the blocking-up of Boc group, N can be held residue acetylation to increase serum stability simultaneously.This type of protecting group is well known to the skilled person.Referring to Greene and Wuts Protective Groups in Organic Synthesis, 1999 (John Wiley and Sons, N.Y).In the time of in peptide is prepared after a while for bi-specific antibody system, they advantageously hold amide from resin cracking to generate corresponding C, to suppress carboxypeptidase activity in body.The synthetic illustrative methods of peptide is disclosed in below in embodiment.

When using bi-specific antibody to carry out pre-targeting, antibody produces or the first binding site of the antigen that associates with target tissue and for haptenic the second binding site on can targeting construct comprising by target tissue.Exemplary hapten includes, but is not limited to HSG and In-DTPA.Raise to the haptenic antibody of HSG be known (for example 679 antibody) and can easily be incorporated in suitable bi-specific antibody (referring to for example U.S. Patent No. 6,962,702,7,138,103 and 7,300,644, about embodiment, be partly incorporated herein by reference).Yet other hapten and the antibody that is incorporated into it is as known in the art and can uses, for example In-DTPA and 734 antibody (for example, U.S. Patent No. 7,534,431, embodiment is partly incorporated herein by reference).

Therapeutic agent

In a plurality of embodiments, therapeutic agent can be used as interferon-antibody DNL as herein described as cytotoxic agent, anti-angiogenic agent, short apoptosis agent, antibiotic, hormone, hormone antagonist, chemotactic factor, medicine, prodrug, toxin, enzyme or other agent ^tMthe complementary therapy of construct.Medicine used can have select free antimitotic agent, antikinase agent, alkylating agent, antimetabolite, antibiotic, alkaloid, anti-angiogenic agent, short apoptosis agent with and the pharmaceutical properties of the group that forms of combination.

Illustrative drug used can comprise 5-fluorouracil, APL (aplidin), azaribine (azaribine), Anastrozole (anastrozole), anthracycline, bendamustine (bendamustine), bleomycin (bleomycin), bortezomib (bortezomib), Bryostatin-1 (bryostain-1), busulfan (busulfan), calicheamycin (calicheamycin), camptothecine (camptothecin), carboplatin (carboplatin), 10-hydroxycamptothecine, carmustine (carmustine), celecoxib (celebrex), chlorambucil (chlorambucil), cisplatin (cisplatin) (CDDP), Cox-2 inhibitor, irinotecan (irinotecan) (CPT-11), SN-38, carboplatin, carat Qu Bin (cladribine), clofarabine (clofarabine), cytarabin (cytosine arabinoside), camptothecin, cyclophosphamide, cytosine arabinoside (cytarabine), dacarbazine (dacarbazine), Docetaxel (docetaxel), dactinomycin (dactinomycin), daunorubicin (daunorubicin), amycin (doxorubicin), 2-pyrrolinyl amycin (2P-DOX), cyano group-morpholinyl amycin, amycin glucosiduronic acid, epirubicin glucosiduronic acid, estramustine (estramustine), table podophyllotoxin (epipodophyllotoxin), estrogen receptor bonding agent, etoposide (etoposide) (VP16), etoposide glucosiduronic acid, etoposide phosphate, floxuridine (FUdR), 3 ', 5 '-O-dioleoyl-FudR (FUdR-dO), fludarabine (fludarabine), flutamide (flutamide), farnesyl-protein transferase inhibitor, tyrosine kinase and Bruton inhibitors of kinases, gemcitabine (gemcitabine), hydroxyurea, idarubicin (idarubicin), ifosfamide (ifosfamide), leunase, lenalidomide (lenolidamide), formyl tetrahydrofolic acid (leucovorin), lomustine (lomustine), chlormethine (mechlorethamine), melphalan (melphalan), purinethol, Ismipur, methotrexate (methotrexate), mitoxantrone (mitoxantrone), mithramycin (mithramycin), mitomycin (mitomycin), mitotane (mitotane), nvelbine (navelbine), nitroso ureas, plicamycin (plicomycin), procarbazine (procarbazine), paclitaxel (paclitaxel), pentostatin (pentostatin), PSI-341, raloxifene (raloxifene), semustine (semustine), streptozotocin (streptozocin), tamoxifen (tamoxifen), taxol (taxol), temozolomide (temazolomide) (aqueous form of DTIC), anti-platinum (transplatinum), Thalidomide (thalidomide), thioguanine, thio-tepa (thiotepa), teniposide (teniposide), topotecan (topotecan), uracil mustard, vinorelbine (vinorelbine), vinblastine (vinblastine), vincristine (vincristine) and vinca alkaloids.

Tyrosine kinase inhibitor used can comprise LFM-A13, Dasatinib (dasatinib), imatinib (imatinib) or AMN107 (nilotinib).

Toxin used can comprise that Ricin, abrin, alpha toxin, saporin, ribonuclease (RNA enzyme) are as ranpirnase, DNA enzyme I, staphylococcus enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, diphtherotoxin, Rhodopseudomonas extracellular toxin and Rhodopseudomonas endotoxin.

Chemotactic factor used can comprise RANTES, MCAF, MIP1-α, MIP1-β and IP-10.

In certain embodiments, can use anti-angiogenic agent, for example angiostatin, bar chalone (baculostatin), canstatin (canstatin), mammary gland silk presses down albumen (maspin), VEGF antibody, anti-PlGF peptide and antibody, anti-angiogene factor antibody, anti-Flk-1 antibody, anti-Flt-1 antibody and peptide, anti-Kras antibody, anti-cMET antibody, anti-MIF (macrophage migration inhibitory factor) antibody, laminin，LN peptide, fibronectin peptide, plasminogen activator inhibitor, tissue inhibitor of metalloproteinase, interferon, interleukin 12, IP-10, Gro-β, thrombospondin, 2ME2, proliferin associated protein, carboxyl acyl aminotriazole (carboxiamidotriazole), CM101, Marimastat (Marimastat), pentosane polysulfate ester, angiopoietin-2, interferon, Antibiotic TAN 420F, PNU145156E, 16K lactotropin fragment, linomide (Roquinimex), Thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, accutin, angiostatin, cidofovir (cidofovir), vincristine, bleomycin, AGM-1470, platelet factor 4 or minocycline (minocycline).

Other useful therapeutic agents can comprise oligonucleotide, particularly preferably for oncogene and oncogene products as the antisense oligonucleotide of bcl-2 or p53.A kind of preferred therapeutic oligonucleotide form is siRNA.

Diagnostic agent

The group that diagnostic agent preferably selects free radionuclide, radiocontrast medium, paramagnetic ion, metal, fluorescent labeling, chemiluminescent labeling, acoustic contrast agent and photosensitizer to form.Described diagnostic agent is well-known, and can use any such known diagnosis agent.The limiting examples of diagnostic agent can comprise radionuclide, for example ¹¹⁰in, ¹¹¹in, ¹⁷⁷lu, ¹⁸f, ¹⁹f, ⁵²fe, ⁶²cu, ⁶⁴cu, ⁶⁷cu, ⁶⁷ga, ⁶⁸ga, ⁸⁶y, ⁹⁰y, ⁸⁹zr, ^94mtc, ⁹⁴tc, ^99mtc, ¹²⁰i, ¹²³i, ¹²⁴i, ¹²⁵i, ¹³¹i, ^154-158gd, ³²p, ¹¹c, ¹³n, ¹⁵o, ¹⁸⁶re, ¹⁸⁸re, ⁵¹mn, ^52mmn, ⁵⁵co, ⁷²as, ⁷⁵br, ⁷⁶br, ^82mrb, ⁸³sr or other gamma emitters, beta emitter or positron emitter.Paramagnetic ion used can comprise chromium (III), manganese (II), ferrum (III), ferrum (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) or erbium (III).Metal contrast agent can comprise lanthanum (III), gold (III), plumbous (II) or bismuth (III).Acoustic contrast agent can comprise liposome as inflation liposome.Radiopaque diagnostic agent can be selected from compound, barium compound, gallium compound and thallium compound.Known extensive multiple fluorescent labeling in this area, includes, but is not limited to Fluorescein isothiocyanate, rhodamine, phycoerythrin (phycoerytherin), phycocyanin, allophycocyanin, o-phthalaldehyde(OPA) and fluorescamine.Available chemiluminescent labeling can comprise luminol, different luminol, aromatics acridinium ester, imidazoles, acridinium salt or oxalate.

Yoke closes technology

In certain embodiments, can be by DNL ^tMconstruct yoke closes in one or more therapeutic agents or diagnostic agent.For example, can be by ¹³¹i is incorporated to the tyrosine of protein or peptide, or is connected to the medicine of the ε amino of lysine residue.Also therapeutic agent and diagnostic agent can be connected to for example SH group of reduction.The known multiple method of preparing the covalently or non-covalently conjugates of therapeutic agent or diagnostic agent and protein or peptide in this area, and can utilize any this type of known method.

Can use isodigeranyl functional cross-link agent as 3-(2-pyridine radicals disulfide group) propanoic acid N-succinyl ester (SPDP) connection therapeutic agent or diagnostic agent.Yu etc., Int.J.Cancer56:244 (1994).The general technology closing for this kind of yoke well known in the art.Referring to for example Wong, CHEMISTRY OF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press1991); Upeslacis etc., " Modification of Antibodies by Chemical Methods, " MONOCLONAL ANTIBODIES:PRINCIPLES AND APPLICATIONS, Birch etc. (volume), 187-230 page (Wiley-Liss, Inc.1995); Price, " Production and Characterization of Synthetic Peptide-Derived Antibodies; " MONOCLONAL ANTIBODIES:PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter etc. (volume), 60-84 page (Cambridge University Press1995).

In some embodiments, chelating agen can be connected to protein or peptide and for example, for chelating therapy agent or diagnostic agent, radionuclide.Exemplary chelating agen includes, but is not limited to DTPA (for example Mx-DTPA), DOTA, TETA, NETA or NOTA.Yoke well known in the art closes and comes with chelating agen the method (referring to for example U.S. Patent No. 7,563,433, embodiment is partly incorporated herein by reference) of connection metal or other parts and protein or peptide.The combination of useful especially metal-chelate comprises 2-benzyl-DTPA and its monomethyl and cyclohexyl analog, can be used from radiological imaging with the diagnosis isotope one that general energy range is 60 to 4,000keV, and described isotope for example ¹²⁵i, ¹³¹i, ¹²³i, ¹²⁴i, ⁶²cu, ⁶⁴cu, ¹⁸f, ¹¹¹in, ⁶⁷ga, ⁶⁸ga, ^99mtc, ^94mtc, ¹¹c, ¹³n, ¹⁵o or ⁷⁶br.When with on-radiation metal as magnesium, ferrum and gadolinium compound tense, identical chelate can be used for MRI.Macro ring chelate can be used with various metals with TETA as NOTA, DOTA together with radioactive metal, more particularly uses with together with the radionuclide of gallium, iridium and copper respectively.Can make described metal-chelate complex highly stable by the size of the metal customization ring for interested.Contain other ring-like chelate as large acyclic polyether, it can be used for stable bond nucleic, for example, in RAIT ²²³ra.

Recently, be disclosed in PET scanning technique and used ¹⁸the method of F labelling, for example, by reacting F-18 and metal or other atoms as aluminum. ¹⁸f-Al conjugates can be connected directly to antibody or for chelation group that can targeting construct at pre-targeted approach labelling as DOTA, NOTA or NETA compound.Described F-18 labelling technique is disclosed in U.S. Patent No. 7,563, in 433.

Therapeutic Method

Various embodiments relate to the method for the treatment of cancer as middle for example, in mammal (comprising the mankind, domestic or companion pet, Canis familiaris L. and cat) experimenter, and it comprises the interferon-antibody DNL to described experimenter's administering therapeutic effective dose ^tMconstruct.Can be by the antibody that is incorporated into antigen on target cell surface or reaction with it of administering therapeutic effective dose simultaneously or sequentially to interferon-antibody DNL ^tMusing of construct supplements.The humanization of the group that preferred other MAb comprise the free MAb composition with reacting below of at least one choosing, chimeric or mankind MAb:CD4, CD5, CD8, CD14, CD15, CD16, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD70, CD74, CD79a, CD80, CD95, CD126, CD133, CD138, CD154, CEACAM5, CEACAM6, B7, AFP, PSMA, EGP-1, EGP-2, carbonic anhydrase IX, PAM4 antigen, MUC1, MUC2, MUC3, MUC4, MUC5ac, Ia, MIF, HM1.24, HLA-DR, tenascin, Flt-3, VEGFR, PlGF, ILGF, IL-6, IL-25, tenascin, TRAIL-R1, TRAIL-R2, complement factor C5, oncogene products or its combination.

Can or sequentially use at least one therapeutic agent and further supplement interferon-antibody DNL by the while ^tMconstruct therapy." CVB " (1.5g/m for example ²cyclophosphamide, 200-400mg/m ²etoposide and 150-200mg/m ²carmustine) for being used for the treatment of the scheme of non-Hodgkin lymphoma.The people such as Patti, Eur.J.Haematol.51:18 (1993).Well-known other the suitable associating chemistry therapeutic schemes of those skilled in the art.Referring to such as Freedman etc., " Non-Hodgkin ' s Lymphomas, " CANCER MEDICINE, the 2nd volume, the 3rd edition, Holland etc. (volume), 2028-2068 page (Lea & Febiger1993).As explanation, the first generation chemotherapy scheme that is used for the treatment of intergrade non-Hodgkin lymphoma (NHL) comprises C-MOPP (cyclophosphamide, vincristine, procarbazine and prednisone) and CHOP (cyclophosphamide, amycin, vincristine and prednisone).Useful second filial generation chemotherapy scheme is m-BACOD (methotrexate, bleomycin, AC, vincristine, dexamethasone and formyl tetrahydrofolic acid), and suitable third generation scheme is MACOP-B (methotrexate, AC, vincristine, prednisone, bleomycin and formyl tetrahydrofolic acid).Other useful medicines comprise phenyl butyrate, bendamustine (bendamustine) and Bryostatin-1.

Can prepare interferon-antibody DNL according to known method ^tMconstruct is to prepare the compositions of pharmaceutically useful, accordingly by interferon-antibody DNL ^tMconstruct with suitable excipient composition pharmaceutically in mixture.Sterile phosphate buffered saline is an example of pharmaceutically suitable excipient.Well-known other the suitable excipient of those skilled in the art.Referring to such as Ansel etc., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, the 5th edition (Lea & Febiger1990), and Gennaro (volume), REMINGTON ' S PHARMACEUTICAL SCIENCES, the 18th edition (Mack Publishing Company1990), with and revised edition.

Can be by interferon-antibody DNL ^tMconstruct preparation is used for intravenous, by for example bolus infusion or continuous transfusion.Preferably, by interferon-antibody DNL ^tMconstruct is through being less than the period of approximately 4 hours and more preferably infusing through being less than the period of approximately 3 hours.For example, initial 25-50mg can be 30 minutes, preferably even transfusion in 15 minutes, and remaining part was through transfusion in 2-3 hour subsequently.Ejection preparation can present by unit dosage forms, for example, in ampoule or multi-dose container, is wherein added with antiseptic.Compositions can be used in suspension, solution or the emulsion form in oiliness or aqueous vehicles, and can comprise preparaton as suspending agent, stabilizing agent and/or dispersant.Alternatively, active component can be powder type, before use with for example aseptic apirogen water reconstruct of suitable carrier.

Can use other pharmaceutical methods to control interferon-antibody DNL ^tMthe persistent period of construct effect.Can by using, polymer be compound or adsorptive hindrance is plain-antibody DNL ^tMconstruct is prepared control delivery formulations.For example, biocompatible polymer comprises the polyanhydride copolymer substrate of poly-(ethylene-altogether-vinylacetate) substrate and stearic acid dimer and decanedioic acid.Sherwood etc., Bio/Technology10:1446 (1992).The rate of release of described substrate depends on interferon-antibody DNL ^tMthe molecular weight of construct, substrate internal interference element-antibody DNL ^tMthe amount of construct and the size of dispersed particle.Saltzman etc., Biophys.J.55:163 (1989); Sherwood etc., the same.Other solid dosage formss are described in Ansel etc., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, the 5th edition (Lea & Febiger1990), and Gennaro (volume), REMINGTON ' S PHARMACEUTICAL SCIENCES, describes in the 18th edition (Mack Publishing Company1990) and revised edition thereof.

Interferon-antibody DNL ^tMconstruct also can to mammal carry out subcutaneous administration or even other parenteral routes use.Described construct is preferably through being less than the period of approximately 4 hours and more preferably infusing through being less than the period of approximately 3 hours.

In more general situation, be applied to interferon-antibody DNL of the mankind ^tMthe dosage of construct will change according to factors such as patient age, body weight, height, sex, general medicine condition of illness and previous medical history.In limiting examples, dosage can be 10 μ g, 20 μ g, 50 μ g, 75 μ g, 100 μ g, 150 μ g, 200 μ g, 250 μ g, 300 μ g, 400 μ g, 500 μ g, 750 μ g, 1mg, 1.5mg, 2mg, 2.5mg, 5mg, 10mg, 20mg, 50mg, 75mg or 100mg.Those skilled in the art will recognize that, if observe toxicity sign, can reduce dosage or stopped treatment.If needed, can repeat administration, for example, continue weekly 4-10 week for twice, continue once in a week 4-10 week, continue 8 weeks once in a week or lasting 4 weeks once in a week.Also can according to maintenance therapy need to be with less frequency administration, for example continue once every two weeks some months, or every month or each season once continue a plurality of months.Alternatively, can every 2 weeks or 3 weeks doses use interferon-antibody DNL ^tMconstruct, repeats at least 3 dosage altogether.Or administered twice construct continues 4-6 week weekly.According to the suitable adjusting to dosage and scheme, dosage regimen optionally repeats with other intervals, and can be by various parenteral route administrations.

In preferred embodiments, interferon-antibody DNL ^tMconstruct can be used for treatment of cancer.The example of cancer includes, but is not limited to carcinoma, lymphoma, glioblastoma multiforme, melanoma, sarcoma and leukemia, myeloma or lymphsystem malignant tumor.The more specifically example of described cancer indicates in lower and comprise: squamous cell carcinoma (for example, epithelium squamous cell carcinoma), Ewing sarcoma, nephroblastoma, astrocytoma, pulmonary carcinoma (comprises small cell lung cancer, nonsmall-cell lung cancer, adenocarcinoma of lung and lung squamous cancer), peritoneal cancer, hepatocarcinoma, gastric cancer (gastric cancer) or gastric cancer (stomach cancer) (comprising human primary gastrointestinal cancers), cancer of pancreas, glioblastoma multiforme, cervical cancer, ovarian cancer, hepatocarcinoma, bladder cancer, hepatoma, hepatocarcinoma, neuroendocrine tumor, medullary thyroid carcinoma, differentiated thyroid carcinoma, breast carcinoma, ovarian cancer, colon cancer, rectal cancer, carcinoma of endometrium or uterus carcinoma, salivary-gland carcinoma, renal carcinoma (kidney cancer) or renal carcinoma (renal cancer), carcinoma of prostate, carcinoma vulvae, anus cancer, carcinoma of penis and head and neck cancer.Term " cancer " (for example comprises primary malignancy cell or tumor, cell does not also move in experimenter's health malignant tumor or the tumor of other positions except initial malignant tumor or knub position) and Secondary cases malignant cell or tumor (for example, deriving from malignant cell or the tumor that metastatic tumor, malignant cell or tumor cell migration arrive the Secondary cases position different from initial knub position).

Other examples of cancer or malignant tumor include, but is not limited to: children acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, adult's (constitutional) hepatocarcinoma, adult's (constitutional) hepatocarcinoma, adult's acute lymphoblastic leukemia, adult's acute myeloid leukemia, adult's Hodgkin lymphoma, adult lymphoid cellularity leukemia, adult's non-Hodgkin lymphoma, Adult Primary hepatocarcinoma, adult soft tissue sarcoma, AIDS associated lymphoma, AIDS associated malignancies, anus cancer, astrocytoma, cancer of biliary duct, bladder cancer, osteocarcinoma, brain stem glioma, the cerebral tumor, breast carcinoma, renal pelvis and carcinoma of ureter, central nervous system's (constitutional) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, child's (constitutional) hepatocarcinoma, child's (constitutional) hepatocarcinoma, children acute lymphoblastic leukemia, children acute myelocytic leukemia, child's brain stem glioma, Cerebellar Astrocytoma in Children. An, child's cerebral astrocytoma, child's extracranial germ cell tumor, child's Hodgkin, study on Hodgkin lymphoma in children, child's hypothalamus and pathways for vision glioma, child's lymphoblastic leukemia, Children Medulloblastoma, Non-Hodgkin Lymphoma in Children, the upper primitive neuroectodermal tumor of child's pinus and curtain, child's primary hepatocarcinoma, Children Rhabdomyosarcoma, child's soft tissue sarcoma, children's vision path and hypothalamus glioma, chronic lymphocytic leukemia, chronic granulocytic leukemia, colon cancer, cutaneous T cell lymphoma, endocrine islet-cell carcinoma, carcinoma of endometrium, ependymoma, epithelial cancer, esophageal carcinoma, Ewing's sarcoma and related neoplasms, exocrine pancreas cancer, extracranial germ cell tumor, Extaagonactal perm celi tumors, cholangiocarcinoma, cancer eye, women with breast cancer, gaucher's disease (Gaucher ' s Disease), carcinoma of gallbladder, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal tumor, germ cell tumor, gestational trophoblastic neoplasms, hairy cell leukemia, head and neck cancer, hepatocarcinoma, Hodgkin lymphoma, hypergammaglobulinemia, hypopharyngeal cancer, intestinal cancer, ophthalmic melanoma, islet-cell carcinoma, islet cells cancer of pancreas, Kaposi sarcoma, renal carcinoma, laryngeal carcinoma, lip and oral cancer, hepatocarcinoma, pulmonary carcinoma, lymphocytic hyperplasia sexually transmitted disease (STD) disease, macroglobulinemia, male breast carcinoma, malignant mesothe, malignant thymoma, medulloblastoma, melanoma, mesothelioma, shift invisible constitutional squamous cell neck cancer, transitivity constitutional squamous cell neck cancer, transitivity squamous cell neck cancer, multiple myeloma, multiple myeloma/plasma cell anything superfluous or useless, myelodysplastic syndrome, myelocytic leukemia, myelomatosis, myeloproliferative disease, nasal cavity and paranasal sinuses, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, nonmelanoma skin cancer, nonsmall-cell lung cancer, invisible constitutional transitivity squamous cell neck cancer, oropharynx cancer, bone/malignant fibrous sarcoma, osteosarcoma/malignant fibrohistiocytoma, osteosarcoma/malignant fibrous histiocytoma of bone, epithelial ovarian cancer, ovarian germ cell tumors, low grade of malignancy ovarian tumor, cancer of pancreas, paraproteinemia, polycythemia vera, parathyroid carcinoma, carcinoma of penis, pheochromocytoma, pituitary tumor, primary central nervous system lymphoma, primary hepatocarcinoma, carcinoma of prostate, rectal cancer, renal cell carcinoma, renal pelvis and carcinoma of ureter, retinoblastoma, rhabdomyosarcoma, salivary-gland carcinoma, sarcoidosis sarcoma, Sezary syndrome, skin carcinoma, small cell lung cancer, carcinoma of small intestine, soft tissue sarcoma, squamous cell neck cancer, gastric cancer, original neuroderm and pinealoma on curtain, t cell lymphoma, carcinoma of testis, thymoma, thyroid carcinoma, renal pelvis and ureteral transitional cell carcinoma, renal pelvis and the ureter cancer of dividing a word with a hyphen at the end of a line, trophoblastic tumor, ureter and renal pelvis cell carcinoma, carcinoma of urethra, uterus carcinoma, sarcoma of uterus, cancer of vagina, pathways for vision and hypothalamus glioma, carcinoma vulvae, Waldenstrom's macroglobulinemia (Waldenstrom ' s Macroglobulinemia), nephroblastoma, and be positioned at tract listed above any other excessively proliferative disease except anything superfluous or useless.

Description and claimed method and composition can be used for treating pernicious or premalignant condition of illness and stop progress to become anything superfluous or useless or malignant tumor state herein, include, but is not limited to those diseases mentioned above.Indication to some extent in the condition of illness that described purposes is anything superfluous or useless or cancer in known or doubtful continuation progress, particularly at non-anything superfluous or useless Growth of Cells, comprise and hypertrophy occurs, change raw or the most hypogenetic time that (summary of described misgrowth situation is referring to Robbins and Angell, Basic Pathology, the 2nd edition, W.B.Saunders Co., Philadelphia, 68-79 page (1976)).

Dysplasia is often the omen of cancer, and mainly in epithelium, finds.It,, for the most unordered a kind of form in non-tumor cell growth, comprises and loses the concordance of independent cell and the structure direction of lost cell.Dysplasia typically occurs in the position that has chronic stimulation or inflammation.Medicable dysplasia disease includes, but is not limited to anhidrotic ectodermal dysplasia, front face dysplasia (anterofacial dysplasia), asphyxiating thoracic dysplasia, atrium-refer to dysplasia, broncho-pulmonary dysplasia, cerebral dysplasia, cervix uteri dysplasia, chondroectodermal dysplasia, cleidocranial dysplasia, congenital ectodermal dysplasia, skull DD (craniodiaphysial dysplasia), the dysplasia of cranium wrist metatarsal, skull metaphysis hypoplasia (craniometaphysial dysplasia), dentine dysplasia, DD, ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia, dysplasia epiphysialis hemimelia, multiple epiphyseal dysplasia, chondrodystrophia congenita punctata, epithelial dysplasia, face-finger (toe)-genital development is bad, familial jawbone fibrous dysplasia, familial white pleat sexual organ dysplasia, Fibromuscular dysplasia, fibrous dysplasia of bone, florid osseous dysplasia, heritability kidney retinal dysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermal dysplasia, lymphopenia thymic hypoplasia, mammary gland dysplasia, lower maxillofacial bone dysplasia, metaphysis dysplasia, Mondini dysplasia, monostotic fibrous dysplasia, mucous epithelium dysplasia, multiple epiphyseal dysplasia, OAVD, oculodentodigital dysplasia, the dysplasia of eye vertebra, tooth source sexual organ dysplasia, jaw dysplasia now, periapical cemental dysplasia, boniness fibrodysplasia, pseudoachondroplasia spondylo epiphyseal dysplasia, retinal dysplasia, depending on every dysplasia, the radially dysplasia of spondylo epiphyseal dysplasia and the ventricles of the brain.

Other medicable tumor diseases in early stage include, but is not limited to hyperplasia of prostate imbalance disease (for example, benign tumor, fibrocyst condition of illness, tissue hypertrophy, polyp intestinal or adenoma and esophagus dysplasia), leukoplakia, seborrheic keratosis, bowen's disease (Bowen ' s disease), farmer's skin sick (Farmer ' s Skin), solar cheilitis and solar keratosis.

In preferred embodiments, the inventive method is for suppressing growth, progress and/or the transfer of cancer, particularly listed cancer above.

Other excessively proliferative diseases, disease and/or condition of illness include, but is not limited to progress and/or the transfer of malignant tumor and associated conditions, and for example leukemia (comprises acute leukemia (for example, acute lymphoblastic leukemia, acute myeloid leukemia (comprises myeloblast property, promyelocyte, Myelomonocyte, monocarpotic cellularity and erythroleukemia)) and (chronic leukemia (for example, chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia))), polycythemia vera, lymphoma (for example, Hodgkin and non-Hodgkin lymphoma), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and entity tumor, include, but is not limited to sarcoma and carcinoma as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, Ewing' s tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, cancer of pancreas, breast carcinoma, ovarian cancer, carcinoma of prostate, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, syringocarcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cancer of biliary duct, choriocarcinoma, spermocytoma, embryonal carcinoma, nephroblastoma, cervical cancer, tumor of testis, pulmonary carcinoma, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma (emangioblastoma), acoustic neuroma, few prominent glioma, meningioma, melanoma, neuroblastoma and retinoblastoma.

In other embodiments, Pegylation DNL ^tMcomplex can be used for treating pathogenic infection organism as the experimenter of antibacterial, virus or fungus.Medicable exemplary fungus comprises Microsporon (Microspomm), trichophyton (Trichophyton), Epidermophyton (Epidermophyton), Sporothrix schenckii (Sporothrix schenckii), Cryptococcus histolyticus (Cryptococcus neoformans), Blastomyces coccidioides (Coccidioides immitis), capsule histoplasma capsulatum (Histoplasma capsulatum), Blastomyces dermatitidis (Blastomyces dermatitidis) or Candida albicans.Exemplary virus comprises HIV (human immunodeficiency virus) (HIV), herpesvirus, cytomegalovirus, rabies virus, influenza virus, human papilloma virus, hepatitis B virus, hepatitis C virus, Sendai virus, feline leukaemia virus, reovirus, poliovirus, the tiny sample virus of serum human, simian virus 40, respiratory syncytial virus, mouse mammary tumor virus, varicella zoster virus, dengue virus, rubella virus, Measles virus, adenovirus, human T-leukemia virus, epstein-Barr virus, muroid leucovirus, mumps virus, vesicular stomatitis virus, sindbis alphavirus, lymphocytic choriomeningitis virus or blue tongue rims.Exemplary antibacterial comprises Bacillus anthracis (Bacillus anthracis), streptococcus agalactiae, legionella pneumophilia, micrococcus scarlatinae, escherichia coli, Diplococcus gonorrhoeae, Neisseria meningitidis, Pn, influenza B haemophilus, syphilis spirillum, lyme disease spirochete, Pseudomonas aeruginosa, Mycobacterium leprae, Bacillus abortus, mycobacterium tuberculosis or mycoplasma.

Test kit

Various embodiments can relate to the test kit that comprises the ingredient that is suitable for treating patient illing tissue.Exemplary kit can contain at least one or multiple interferon-antibody construct as described herein.If contain the compositions of using ingredient, be not configured to by digestive tract and send, for example oral delivery, can comprise in test kit and can pass through the device of some other approach delivery of agents box ingredients.The confession of one type is used if non-intestinal delivery apparatus is for for entering compositions injection the syringe in subject.Also can use suction apparatus.In certain embodiments, can contain the precharging type syringe of sterile liquid formulations or lyophilized formulations or the form of automatic injection pen provides therapeutic agent.

Each ingredient of test kit can be packaging together or be divided in two or more containers.In some embodiments, container can be the phial of the sterile freeze-drying preparation that contains the compositions that is suitable for reconstruct.Test kit also can comprise the buffer that one or more are suitable for reconstruct and/or dilute other reagent.Other available containers include, but is not limited to capsule, dish, box, pipe etc.Test kit ingredient can and keep aseptic in container inner packing.The ingredient that another one can comprise is used the description of test kit for people.

Embodiment

Provide following examples for illustrating but do not limit claim of the present invention.

Embodiment 1.C _h3-AD2-IgG expression vector

PdHL2 mammalian expression vector has been used to express restructuring IgG (Qu etc., Methods2005,36:84-95).Producing a kind of plasmid shuttle vector assists and changes any IgG-pdHL2 carrier into C _h3-AD2-IgG-pdHL2 carrier.Use pdHL2 carrier as template and use following oligonucleotide primers by pcr amplification Fc (C _h2and C _h3domain) gene:

Fc BglII is left

AGATCTGGCGCACCTGAACTCCTG(SEQ?ID?NO：90)

Fc Bam-EcoRI is right

GAATTCGGATCCTTTACCCGGAGACAGGGAGAG(SEQ?ID?NO：91)。

Amplimer is cloned in pGemT PCR cloning vehicle (Promega).Use Xba I and Bam HI shears Fc Insert Fragment in pGemT and with by using Xba I and Bam HI to digest h679-Fab-AD2-pdHL2 (Rossi etc., Proc Natl Acad Sci USA2006,103:6841-6) the AD2-pdHL2 carrier prepared connects to generate shuttle vector Fc-AD2-pdHL2.For IgG-pdHL2 expression vector is converted into C _h3-AD2-IgG-pdHL2 expression vector, shears the BsrG I/Nde I restricted fragment of 861bp get off and uses the BsrG I/Nde I restricted fragment displacement of shearing from the 952bp of Fc-AD2-pdHL2 carrier from the former.Below for having generated and for generation of the C of recombinant humanized IgG-AD2 module _h3the part list of-AD2-IgG-pdHL2 expression vector:

C _h3-AD2-IgG-hA20 (anti-CD20)

C _h3-AD2-IgG-hLL2 (anti-CD22)

C _h3-AD2-IgG-hL243 (anti-HLA-DR)

C _h3-AD2-IgG-hLL1 (anti-CD74)

C _h3-AD2-IgG-hR1 (anti-IGF-1R)

C _h3-AD2-IgG-h734 (anti-indium-DTPA).

Embodiment 2.C _hthe generation of 3-AD2-IgG

stable C _h transfection and the selection of the thin bag of 3-AD2-IgG secretion system

The all growths in hybridoma SFM (Invitrogen, Carlsbad CA) of all cell line.By using Sal I digestion with restriction enzyme and entering Sp2/0-Ag14 (2.8 * 10 by electroporation (450 volts, 25 μ F) transfection ⁶individual cell) by C _h3-AD2-IgG-pdHL2 carrier (30 μ g) linearisation.The gene that described pdHL2 carrier comprises dihydrofolate reductase, allows to use methotrexate (MTX) to carry out Immune Clone Selection and gene amplification.

After transfection, cell is selected to transgene clone in bed board the culture medium that containing 0.2 μ M MTX on 96 orifice plates.By using each C cloning of sandwich ELISA screening of 96 hole microtitration plates of specificity antiidiotype MAb coating _h3-AD2-IgG productivity ratio.By the conditionality media transfer of inferring clone in micro-plate hole, and the anti-IgG F of the goat of using horseradish peroxidase yoke to close (ab ') ₂(Jackson ImmunoResearch Laboratories, West Grove, PA) detects fusion rotein.By provide the hole amplification of highest signal and finally for the production of.

c _h the generation of 3-AD2-IgG module and purification

In order to produce fusion rotein, with 2 * 10 ⁵the density of individual cell/ml is sowed cell and at 37 ℃ and 5%CO in roller bottle is cultivated ₂under in roller bottle incubator incubation until cell survival rate drop to lower than 25% (approximately 10 days).By centrifugal, culture broth is clarified, filtered and by ultrafiltration and concentration to 50 times.For purification C _h3-AD2-IgG module, is loaded to concentrated supernatant on protein A (MAB Select) affinity column.Use PBS that this post is washed till to baseline and uses 0.1M glycine (pH2.5) by this fusion rotein eluting.

The Fab that embodiment 3. is connected with DDD from the AD of Multiple Antibodies and the generation of IgG fusion rotein

Use the technology described in previous embodiment, the IgG shown in structure table 6 and Fab fusion rotein and be incorporated in DNL construct.Antigenic binding property and DNL construct that described fusion rotein has retained parental antibody represent be incorporated to antibody or the antigen-binding activity of antibody fragment.

The fusion rotein that table 6. comprises IgG or Fab

The generation of the DDD module of embodiment 4. based on interferon (IFN)-α 2b

structure for the IFN-α 2b-DDD2-pdHL2 that expresses at mammalian cell

By the cDNA sequence of pcr amplification IFN-α 2b, obtain the sequence that comprises following characteristics, wherein XbaI and BamHI are restriction site, signal peptide is IFN-α 2b natural signals peptide, and 6His is hexahistidine tag: XbaI---signal peptide---IFN α 2b---6His---BamHI (6His is open with SEQ ID NO:92).Gained secretory protein is comprised of the IFN-α 2b that merges the polypeptide of following sequence at its C end:

KSHHHHHHGSGGGGSGGGCGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：93)。

Using total length mankind IFN α 2b cDNA clone (the human cloned catalogue #HORF01 clone of InVitrogen Ultimate ORF ID IOH35221) is that template and following oligonucleotide are that primer is realized pcr amplification:

IFNA2Xba I is left

TCTAGACACAGGACCTCATCATGGCCTTGACCTTTGCTITACTGG(SEQ?ID?NO：94)

IFNA2BamHI is right

GGATCCATGATGGTGATGATGGTGTGACTTTTCCTTACTTCTTAAACTTTCTTGC(SEQ?ID?NO：95)

Pcr amplification primer is cloned in pGemT carrier.Be prepared as follows for connecting the DDD2-pdHL2 mammalian expression vector of IFN-α 2b.Use Xba I and Bam HI by C _h1-DDD2-Fab-hMN-14-pdHL2 (Rossi etc., Proc Natl Acad Sci USA2006,103:6841-6) vector digestion, removes all Fab gene orders and still retains DDD2 coded sequence.Use Xba I and Bam HI that IFN-α 2b amplimer is sheared and is connected on DDD2-pdHL2 carrier to generate expression vector IFN-α 2b-DDD2-pdHL2 from pGemT.

the mammalian cell expression of IFN-α 2b-DDD2

Use Sal I by IFN-α 2b-DDD2-pdHL2 digestion and linearisation, and be stably transfected into (referring to U.S. Patent application 11/877,728, embodiment is partly incorporated herein by reference) in Sp/ESF myeloma cell by electroporation.Utilize ELISA find two clones have can detection level IFN-α 2b.One of these two clones, called after 95 can adapt to and grow in serum-free medium in the situation that significantly not reducing productivity ratio.With by five weeks, this clone being increased with the MTX of 0.1 to 0.8 μ M progressive concentration.In this stage, by restriction, dilute by its sub-clone and by sub-clone (95-5) amplification of high yield.Using business rIFN-α 2b (Chemicon IF007, lot number 06008039084) is reference material, and the estimated output that is grown in the 95-5 in shaking flask is 2.5mg/L.

purification IFN-α 2b-DDD2 in the batch culture of growing from roller bottle

Clone 95-5 is increased 34 in the roller bottle that contains 20L serum-free hybridoma SFM and 0.8 μ M MTX altogether and make it to arrive cultivation in latter stage.Culture broth is processed and passed through as follows immobilization metal affinity chromatography (IMAC) by IFN-α 2b-DDD2 purification.By the centrifugal supernatant that makes, clarify, 0.2 μ M filters, and diafiltration is to 1X binding buffer liquid (10mM imidazoles, 0.5M NaCl, 50mM NaH ₂pO ₄, pH7.5) in, be concentrated into 310mL, add Tween20 to reach ultimate density 0.1%, and be loaded on 30mL Ni-NTA post.After sample loading, use successively the 0.02%Tween20 in 1X binding buffer liquid of 500mL and 30mM imidazoles, 0.02%Tween20,0.5M NaCl, the 50mM NaH of 290mL ₂pO ₄(pH7.5) column scrubber.Use 250mM imidazoles, 0.02%Tween20,150mM NaCl, the 50mM NaH of 110mL ₂pO ₄(pH7.5) by product eluting.Purification obtains about 6mg IFN α 2b-DDD2.

the generation of IFN-α 2b-DDD2 in escherichia coli

Also IFN-α 2b-DDD2 can be fermented at expression in escherichia coli by microorganism with soluble protein form.Use IFN-α 2b-DDD2-pdHL2 DNA coded sequence to be passed through to pcr amplification for template.Use Nde I and Xho I restriction site that this amplimer is cloned in pET26b coli expression carrier.By using 100 μ m IPTG to carry out induction in 12 hours to LB shaking flask at 18 ℃, protein is expressed in cell in BL21pLysS host cell.As mentioned above by IMAC by solubility IFN-α 2b-DDD2 purification from cell lysates.

Embodiment 5. comprises and is connected to C _hthe generation of the DNL conjugates of four IFN-α 2b-DDD2 parts of 3-AD2-IgG

Be prepared as follows to comprise and be connected to C _h3the DNL complex (Fig. 1) of four IFN-α 2b-DDD2 parts of-AD2-IgG.Concise and to the point, by selected C _hthe IFN-α 2b-DDD2 combination of 3-AD2-IgG and about two molar equivalents, and after adding 1mM EDTA and 2mM reduced glutathion (GSH), this mixture is reduced in ambient temperature overnight under gentle condition.Add oxidized form of glutathione to 2mM and this mixture is placed in to room temperature 12-24 hour again.By this DNL conjugates of protein A affinity column purification.Prepare four kinds of these type of DNL conjugatess that are designed to 20-2b, 22-2b, hR1-2b and 243-2b, what respectively comprise four copies is anchored to respectively C _h3-AD2-IgG-hA20 (thering is CD20 specificity), C _h3-AD2-IgG-hLL2 (thering is CD22 specificity), CH3-AD2-IgG-hR1 (thering is IGF-1R specificity) and C _hiFN-α 2b on 3-AD2-IgG-hL243 (thering is HLA-DR specificity).The 20-2b that the IFN-α 2b-DDD2 that uses SE-HPLC to produce mammal (m) or escherichia coli (e) generates analyzes and shows, every kind demonstrates a holdup time main peak (not shown) consistent with the covalent complex being comprised of IgG and 4 IFN-α 2b groups.In other three kinds of IFN-IgG conjugatess, observe similar SE-HPLC overview.

The external activity of embodiment 6.IFN-IgG conjugates.

The reporter gene of use based on cell, virus protection and lymphoma proliferation assay compare the external IFN α biological activity of 20-2b and 2 doses of PEGASYS of business Pegylation IFN α and PEG-intron.The test kit of use based on cell determined activity specific, and it utilizes with the transgenic mankind premonocyte system (Fig. 2 A-2D) that is blended in the reporter gene of interferon irritant reaction element.The activity specific of 20-2b (5300IU/pmol) is greater than PEGASYS (170IU/pmol) and PEG-intron (3400IU/pmol) (Fig. 2 A).734-2b, the 1R-2b and five kinds of other MAb-IFN α constructs (data are not shown) that to be similar to the method for 20-2b, produce, all show similar activity specific (4000-8000IU/pmol), show to generate the concordance (Fig. 2 A) of the DNL method of this class formation.There is the effect that four IFN α 2b groups contribute to strengthen MAb-IFN α.After to the normalization of IFN α equivalent, activity specific/IFN α is higher about 10 times than PEGASYS, and only low about 2 times than PEG-intron.

In virus protection algoscopy, MAb-IFN α, PEGASYS and PEG-intron are compared in vitro, show that it is also the IFN α 2b antiviral activity (Fig. 2 B) of PEGASYS10 activity specific doubly that MAb-IFN α reserved category is similar to PEG-intron.

IFN α 2b can have direct antiproliferative or cytotoxic effect to some tumor cell lines.In vitro in proliferation assay the extremely sensitive Burkitt lymphoma cell line of IFN α (Daudi) is carried out to the measurement (Fig. 2 C) of 20-2b activity.2 doses of each IFN α are in vitro with efficient (EC ₅₀=4-10pM) effectively suppress (> 90%) Daudi.Yet, 20-2b (EC ₅₀=0.25pM) than non-targeted MAb-IFN α construct more potent about 30 times.(EC under quite high concentration ₅₀> 10nM), the anti-CD20MAb of the parent of 20-2b has In Vitro Anti proliferation activity (Rossi etc., 2008, Cancer Res68:8384-92) in multiple lymphoma cell line (comprising Daudi).Also the external activity that uses Jeko-1 assessment 20-2b, Jeko-1 is for to have the lymphoma mantle cell cell line (Fig. 2 D) compared with hyposensitivity to IFN α and anti-CD20.Jeko-1 is only medium sensitivity for the anti-CD20MAb of parent, at EC ₅₀while approaching 1nM, there is 10% maximum inhibition (I _max).As shown in 734-2b, Jeko-1 (I _max=43%; EC ₅₀=23pM) than Daudi (I _max=90%; EC ₅₀=7.5pM) less to IFN α 2b response.Than 734-2b, 20-2b suppresses Jeko-1 (I in larger degree _max=65%) and show two-phase dose-response curve (Fig. 2 D).Under < 10pM, observe the low concentration response owing to IFN α 2b activity, it is in I _max=43% is stable, similar to 734-2b.More than 100pM, can obviously observe high concentration response, wherein I _maxreach 65%.The low concentration IFN α 2b response (EC of 20-2b ₅₀=0.97pM) than 734-2b effective 25 times, the result of Daudi is similar with using.

The increase that the combination of parent's anti-CD 20 antibodies and 734-2b (dimension trastuzumab+734-2b) is measured to illustrate 20-2b effect whether owing to CD20 and the conduction of IFN alpha signal add and/cooperative effect.The dose response curve of v-mab+734-2b is most of similar to independent 734-2b, except when the > 1nM, and the former but not the latter's inhibition reinforcement.These results show that MAb targeting causes the EC of 20-2b ₅₀lower, and its larger I _maxobvious adding and activity owing to IFN α 2b and the conduction of CD-20 signal.The effect of CD20 signal conduction only responds (EC in the high concentration of 20-2b ₅₀=0.85nM) in obviously, its with to v-mab (EC ₅₀=1.5nM) response is parallel.The two-phase dose-response curve of v-mab+734-2b is not obvious, because these two responses are overlapping.Yet, under the concentration of > 1nM, have obvious additive effect.The I of 20-2b _max(65%) than IFN α 2b (I _max=43%) and dimension trastuzumab (I _max=10%) add and response high, show may have cooperative effect between the effect of IFN α 2b and v-mab (dimension trastuzumab).

ADCC is active

IFN α can strengthen ADCC activity by activation NK cell and macrophage, and it is the basic role mechanism (MOA) of anti-CD20 immunotherapy.We use peripheral blood lymphocytes (PBMC) action effect cell under two kinds of NHL cell lines, to compare the ADCC of 20-2b and v-mab.Use as one man shows that from the replication method of a plurality of donors' PBMC 20-2b has the ADCC of enhancing than v-mab, as shown in about Daudi and Raji cell (Fig. 4 A).This effect also shows 22-2b (a kind of MAb-IFN α that comprises anti-CD22MAb epratuzumab), and it shows medium ADCC (Carnahan etc., 2007, Mol Immunol44:1331-41).

CDC is active

CDC is considered to the anti-CD20MAb of the I type important MOA of (comprising v-mab and Rituximab).Yet, lack this function (Cardarelli etc., 2002, Cancer Immunol Immunother51:15-24) take in the II type MAb that tositumomab is representative, although it still has lymphoma activity.Different from v-mab, 20-2b does not demonstrate CDC activity (Fig. 4 B) in vitro.These results and other are based on C _hthe DNL structure of 3-AD2-IgG-v-mab module is consistent, and it may disturb by steric hindrance, makes wherein complement fixation be subject to obvious damage (Rossi etc., 2008, Cancer Res68:8384-92).

The pharmacokinetics of embodiment 7.20-2b (PK) is analyzed.

In male Swiss-Webster mice, evaluate 20-2b pharmacokinetics (PK) character and with PEGASYS, PEG-intron and α 2b-413 (the Pegylation IFN that uses DNL to prepare, referring to U.S. Patent application sequence No.11/925,408) compare.Utilize ELISA according to manufacturer's explanation, under a plurality of time, to measure the concentration of IFN-α in blood serum sample.In brief, according to the mankind IFN-α reference material providing in test kit, blood serum sample is suitably diluted.Be incorporated into the antibody capture interferon of micro titer plate well.Then use second antibody to show the interferon of combination, after adding tetramethyl benzidine (TMB), by yoke, be bonded to the quantitative interferon of anti-secondary antibody of horseradish peroxidase (HRP).Under 450nm, read plate.Fig. 3 presents the result that PK analyzes, and it shows that 20-2b is than the significantly slower elimination of other agent and longer serum demurrage.In the situation that injected dose is 210pmol, the pharmacokinetics serum half-life (take hour) that calculates gained is 8.0 hours (20-2b), 5.7 hours (α 2b-413), 4.7 hours (PEGASYS) and 2.6 hours (PEG-intron).Eliminating speed (1/h) is 0.087 (20-2b), 0.121 (α 2b-413), 0.149 (PEGASYS) and 0.265 (PEG-intron).Calculate the MRT of gained _0.08→ ∞ (hour) be 22.2 (20-2b), 12.5 (α 2b-413), 10.7 (PEGASYS) and 6.0 (PEG-intron).Because pharmacokinetic parameter is decided by the character of this complex but not independent antibody or cytokine more, so the PK characteristic of expection cytokine-DNL complex can be common to other cytokines parts and antibody moiety and be not limited to specific 20-2b construct discussed above.

The activity in vivo of embodiment 8.20-2b

Serum stability

20-2b stablizes (not shown) at 37 ℃ in serum human (>=10 days) or whole blood (>=6 days).Use bispecific ELISA algoscopy to determine the concentration of 20-2b complex.In the period of algoscopy, in whole blood or serum, serum 20-2b level there is no detectable variation.

The in vitro effect of the anti-lymphoma cell from human whole blood of 20-2b

We have compared 20-2b, v-mab, 734-2b or v-mab+734-2b and under in vitro configuration, from whole blood, have eliminated the ability (Fig. 5) of lymphoma or normal B cell.The therapeutic efficiency of naked anti-CD20MAb is considered to by apoptosis or growth retardation, ADCC and the CDC (Glennie etc., 2007, Mol Immunol44:3823-37) of three kinds of mechanism of action (MOA) realization-signal conduction induction.In this algoscopy, v-mab can adopt all three kinds of MOA, yet based on results of in vitro studies, 20-2b can conduct with signal and strengthen ADCC but not CDC by potential land productivity.In this short-run model, the IFN α 2b group of 20-2b and 734-2b can directly act on tumor cell, and the ADCC that strengthens v-mab is active, and may have some immunostimulations.Yet, in biduous isolated measuring method, realize the activation of the congenital and adaptive immune system of the alpha mediated full spectrum of the IFN that may occur in vivo.

Under 0.01nM, 20-2b (60.5%) consumes significantly more Daudi cell (Fig. 5) than v-mab (22.8%), 734-2b (38.6%) or v-mab+734-2b (41.7%).Under 0.1nM, 20-2b and v-mab+734-2b consume the Daudi (88.9%) of similarity degree, and it is than v-mab (82.4%) or 734-2b (40.7%) many (Fig. 5).Under 1nM, except 734-2b (55.7%), each agent consumes the Daudi (Fig. 5) of > 95%.The difference tool statistical significance (P < 0.01) that each is indicated.

For IFN α 2b and v-mab, Ramos is not as Daudi sensitivity.The effect of 734-2b is only medium, causes the consumption < 20% (Fig. 5) of Ramos under each concentration.0.01 and 0.1nM under, 20-2b consumes more Ramos than v-mab+734-2b, itself then than v-mab, eliminate more cell (Fig. 5).Under 1nM, except 734-2b, all processing all obtain similar Ramos and consume (75%) (Fig. 5).The difference tool statistical significance (P < 0.02) that each is indicated.

As shown in about 734-2b, in this algoscopy, IFN α 2b can not consume normal B cell separately.Under these low concentrations, 20-2b, v-mab and v-mab+734-2b show the dose response consumption of similar B cell separately, and it is significantly less than the consumption of Daudi or Ramos.All processing can not cause significant t cell depletion (data are not shown).

Effect in the body of 20-2b in SCID mice

One of mouse model is restricted to the utmost point hyposensitivity of muroid cell to mankind IFN α 2b.The wholistic therapy benefit that 20-2b can realize in the mankind can comprise the congenital and adaptive immunity of enhancing.After understanding these restrictions, we have studied lymphoma effect in the body of 20-2b anti-dissemination Burkitt lymphoma model in SCID mice.Originally we tested the early stage Daudi model of hypersensitivity (Fig. 6 A).In inoculation one day after, to each group, use 20-2b, v-mab or the 734-2b of single low dosage.The v-mab of the 0.7pmol of single dose (170ng) (dimension trastuzumab) or 734-2b are compared with saline, v-mab causes survival rate to significantly improve (P < 0.0001), but uncorrelated MAb-IFN α contrast 734-2b is without this performance (Fig. 6 A).This raising is medium, and the intermediate value time-to-live (MST) is increased to 34 days of v-mab for 27 days from saline.Yet the 20-2b of the 0.7pmol of single dose (170ng) makes MST improve more than 100 days (P < 0.0001) (Fig. 6 A) than saline control and v-mab group.The research stopped after 19 weeks, now 7 long-term survivorses (LTS) in 0.7pmol20-2b processed group was carried out to necropsy, did not find visible disease indication (curing) (Fig. 6 A).Even if it should be noted that the 20-2b of the 0.07pmol (17ng) of lowest dose level not only makes MST double (Fig. 6 A).

Next, we assess the effect of 20-2b in having more Daudi model in challenging late period, wherein make mice before processing, form larger substantially tumor load (Fig. 6 B).After tumor inoculation seven days, to each group, use 20-2b, v-mab, 734-2b or the PEGASYS of single low dosage (0.7,7.0 or 70pmol).The MST of saline control mice is 21 days (Fig. 6 B).The PEGASYS of maximum dose level (70pmol) or 734-2b have the Pk (than restructuring IFN α 2b) of enhancing separately, but targeting is not in tumor, and they double MST (42 days; P < 0.0001) (Fig. 6 B).Use 20-2b to produce the result (38.5 day) (Fig. 6 B) similar to the PEGASYS of maximum dose level (70pmol) or 734-2b lower processing of 100 times of low dosages (0.7pmol).Use 20-2b 10 times of low dosages (7pmol) lower process cause than use the PEGASYS of 70pmol or survival rate that 734-2b processes significantly improve (80.5 days, 20%LTS) (P < 0.0012) (Fig. 6 B).Under tested maximum dose level (70pmol), 20-2b is increased to MST > 105 days and has 100%LTS (Fig. 6 B).In tumor model, we had previously proved that v-mab can increase the survival with the mice of Daudi under relatively low dosage (3.5pmol) in early days, and higher dosage produces LTS.Yet in this late tumor model, but the v-mab that single dose is 70pmol only has significantly medium effect (MST=24 days, P=0.0001) (Fig. 6 B) for survival.

We measure 20-2b subsequently in having more challenging model, and this model is more insensitive for the direct rejection ratio Daudi of IFN α, and more do not respond for v-mab immunotherapy.Raji is little approximately 1000 times for the direct acting sensitivity of IFN α 2b than Daudi.Yet, Raji have the CD20 antigen density similar with Daudi (Stein etc., 2006, Blood108:2736-44) and to v-mab response, but than the response of Daudi significantly little a lot (Goldenberg etc., 2009, Blood113,1062-70).At tumor inoculation begin treatment after five days, in Raji model, study late the effect (Fig. 7 A) of 20-2b.Through two circumferential each groups, use totally 6 injections (each 250pmol).With respect to saline, 734-2b does not increase survival (MST=16 days), this with Raji to the insensitivity of IFN α consistent (Fig. 7 A).With respect to saline, V-mab significantly increases survival (MST=26 days, P < 0.000l) (Fig. 7 A).20-2b is than other all processing more effectively (MST=33 days, P < 0.0001) (Fig. 7 A).

Finally, we have studied the effect (Fig. 7 B) of 20-2b in NAMALWA situation, NAMALWA is that a kind of direct effect to IFN α has hyposensitivity, low approximately 25 times than the CD20 antigen density of Daudi or Raji, and being considered to resist CD20 immunotherapy has the human lymphoma of resistance (Stein etc., 2006).To each group, use 20-2b or the 734-2b of totally 6 dosage (each dosage 250pmol).To another group, use the v-mab of totally 7 dosage (each dosage 3.5nmol).By the group of saline treatment, there is the MST (Fig. 7 B) of 17 days.By the group that 734-2b processes, there is significantly still the very survival of moderate and improve (MST=20 days, P=0.0012) (Fig. 7 B).20-2b (MST=34 days) than 734-2b (P=0.0004) and v-mab (MST=24 days, P=0.0026) more effective, and v-mab gives dose ratio 20-2b high 14 times (Fig. 7 B).

Conclusion

Acquired results clearly shows immune cell factor to be used separately or to combine use more potent and effective than any agent with anti-CD20MAb targeting IFN α.The MAb target tumor of IFN α make can more low-frequency scheme to independent agent, lower or eliminate the relevant side effect of IFN therapy, and the effect that obtains significantly strengthening.In addition, the IFN α of institute's targeting can bring out the directed immunoreation of acute tumor, and may be by the pleiotropy of congenital and adaptive immunity be stimulated and arouses immunological memory (Belardelli etc., 2002, Cytokine Growth Factor Rev12:119-34).By chemical yoke symphysis, become other groups of MAb-IFN α to disclose some potential clinical benefits (Pelham etc., 1983, the Cancer Immunol Immunother15:210-16 of this type of construct; Ozzello etc., 1998, Breast Cancer Res Treat48:135-47).The restructuring MAb-IFN α that comprises muroid IFN α and anti-HER2/neu MAb shows the potent inhibition to transgenic (HER2/neu) muroid B cell lymphoma in having immunocompetent mice; and can bring out protective adaptation immunoreation (Huang etc. according to immunological memory; 2007, J Immunol179:6881-88).

We expect that 20-2b therapy comprises stimulation local recruitment and the activation of the panimmunity cell of NK, T4, T8 and dendritic cell, cause the cytotoxicity and the ADCC that strengthen, and the directed immunological memory of induced tumor potentially.Yet for mankind IFN α 2b, the relative mankind's cell polar of the sensitivity the earth of muroid cell reduces (about 4log) (Kramer etc., 1983, J Interferon Res3:425-35; Weck etc., 1981, J Gen Virol57:233-37).Therefore in above-mentioned mouse model body, in research, even if having, be, also that the lymphoma activity of considerably less 20-2b is attributable to the activation of IFN α 2b to mouse immune reaction.On the contrary, death is mainly due to the direct effect of IFN α 2b to lymphoma cell.

We have shown that 20-2b can strengthen ADCC, and this may be the most important MOA of anti-CD20 immunotherapy.Yet, because mankind IFN α 2b is only the very weak stimulant of Rat host immune effector cell, so may not can realize the ADCC that IFN α strengthens as in the mankind.Even if having these limitation, result still shows that 20-2b can be efficient lymphoma agent in body, shows than the effect of many 100 times of v-mab or non-targeted property MAb-IFN α in IFN α sensitivity Daudi model.Even IFN α direct effect relative insensitivity (Raji/NAMALWA) or antagonism CD20 immunotherapy are had in the lymphoma model of resistance (NAMALWA), 20-2b still demonstrates than v-mab or the non-targeted higher effect of MAb-IFN α.

The fusion of IFN α 2b to v-mab is by extending circulation time and allowing cancer target to strengthen effect in its body.In Daudi model, show the treatment significance of Pk, wherein slow PEGASYS removing is better than the PEG-intron than very fast removing, although the latter has higher activity specific (data are not shown).20-2b has significantly larger effect than PEGASYS or 734-2b, shows for its more excellent effect and effect, to play decisive role by anti-CD20MAb targeting lymphoma.Unexpectedly, the impact of targeting is also very remarkable in algoscopy even in vitro.In proliferation experiment, only allow to be conducted lymphoma is suppressed by signal in vitro, with respect to non-targeted MAb-IFN α, no matter the in the situation that of v-mab alone or in combination, 20-2b all shows activity under the concentration of relatively low 25 times.In vitro configuration allows to relate to three kinds of all anti-CD20MOA.Active even without CDC, no matter alone or in combination in the situation that, 20-2b more effectively consumes lymphoma than IFN α or v-mab from blood, has shown the importance of targeting.And the impact of MAb targeting is more or less unexpected in vitro/vitro study, because MAb, effector and target cell are all limited in whole experiment.During our expection is tested in the body of human patients, 20-2b will have larger substantially impact.

The IFN α 2b of 20-2b and v-mab ingredient can add obviously and or synergism, to facilitate the effect of its enhancing.In-vitro multiplication algoscopy shows to have at least one addition, and it is better than using separately the result of any dose to confirm by obtain associating v-mab and 734-2b in vitro study.This can be active or realize in vitro while combining with 734-2b by increasing ADCC as the v-mab of a part of 20-2b, however in vitro in proliferation assay ADCC still tool is not functional, show to exist other mechanism.The signal transduction causing in conjunction with the CD20 of v-mab can strengthen IFN alpha signal, causes the effect strengthening.Alternatively, in conjunction with the internalization/downward that can prevent I type IFN receptor, produce longer time and effective IFN α inducement signal with v-mab (it is a kind of MAb of slow internalization).

Embodiment 9. (Fab) ₂the DNL construct of-interferon-λ 1 shows effective biological activity to the cell of institute's targeting

General introduction and foreword

Interferon lambda 1 (IFN-λ 1) is the type iii interferon that is recently described to II type cytokines family member, about antiviral and anti-tumor activity and immune system, has treatment potentiality.IFN-λ, as I type IFN, (comprise IFN-α and IFN-β), by JAK/STAT approach triggering signal, transduce, activation (the Witte etc. that comprise the phosphorylation of the kinase whose activation of JAK1 and TYK2, stat protein and the transcription complex of the gene factor 3 that IFN stimulates, 2010, Cytokine Growth Factor Rev21:237-51; Zhou etc., 2007, J Virol81:7749-58).

Main Differences between III type IFN and I type IFN is the distribution of its corresponding receptor complex.IFN-α/β is carried out signal conduction by the I type interferon receptors of two kinds of wide expression, and this has caused the general toxicity relevant to IFN-α/β therapy (Pestka, 2007, J Biol Chem282:20047-51) at least partly.By contrast, IFN-λ carries out signal conduction by the allos dimerization receptor complex that comprises IFN-λ receptor 1 (IFN-λ R1) and IL-10 receptor 2 (IL-10R2).And IL-10R2 generally expresses in hematopoietic cell and non-hematopoietic cell, IFN-λ R1 has height-limited expression pattern, wherein in epithelial cell, melanocyte and hepatocyte, level is the highest, and in primary central nervous system cell the minimum (Wolk etc. of level, 2005, Genes Immun6:8-18).Although haematogenic immunity cellular expression IFN-λ is R1, it is because the short splice variant of IFN-λ R1 that secretion can suppress the effect of IFN-λ 1 represents the response of IFN-λ impaired (Witte etc., 2009, Genes Immun10:702-14).The toxicity that the finite response of neuronal cell and immunocyte also impels IFN-λ is than I type IFN decrease (Witte etc., 2009, Genes Immun10:702-14).

IFN-λ shows the cytokine similar architectural feature relevant with IL-10, but represents I type IFN class antiviral and antiproliferative activity (Witte etc., 2010, Cytokine Growth Factor Rev21:237-51; Ank etc., 2006, J Virol80:4501-9; Robek etc., 2005, J Virol79:3851-54).For example, research has shown that IFN-λ 1 and IFN-λ 2 can reduce the cytopathic effect of virus replication or various viruses, and described virus comprises DNA viruses, such as hepatitis B virus (Robek etc., 2005, J Virol79:3851-54; Doyle etc., 2006, Hepatology44:896-906) with herpes simplex types 2 virus (Ank etc., 2006, J Virol80:4501-9); Sense single stranded rna virus, such as encephalomyocarditis virus (EMCV) (Sheppard etc., 2003, Nat Immunol4:63-68) and hepatitis C virus (HCV) (Robek etc., 2005, J Virol79:3851-54; Doyle etc., 2006, Hepatology44:896-906); Negative adopted single strand RNA virus, such as vesicular stomatitis virus (Kotenko etc., 2003, Nat Immunol6:69-77; Pagliaccetti etc., 2008, J Biol Chem283:30079-89) and influenza A virus (Jewell etc., 2010, J Virol84:11515-22); And diplornavirus, such as rotavirus (Pott etc., 2011, PNAS USA108:7944-49).IFN-λ 3 is accredited as the key cytokines (Ge etc. in HCV infection according to genetic research, 2009, Nature461:399-401), and there are the effective active (Dellgren etc. for EMCV, 2009, Genes Immun10:125-31).

Also in several human cancer cell line, determined the antiproliferative activity of IFN-λ, comprise neuroendocrine carcinoma BON1 (Zitzmann etc., 2006, BBRC344:1334-41), glioblastoma multiforme LN319 (Meager etc., 2005, Cytokine31:109-18), immortalization keratinocyte HaCaT (Maher etc., 2008, Cancer Biol Ther7:1109-15), melanoma F01 (Guenterberg etc., 2010, Mol Cancer Ther9:510-20) and esophageal carcinoma TE-11 (Li etc., 2010, Eur J Cancer46:180-90).In animal model, IFN-λ is by congenital and the reaction induced apoptosis of tumor cells of adaptive immunity and elimination, this shows that the local delivery of IFN-λ may be available strategy (Numasaki etc., 2007, the J Immunol178:5086-98 in human malignancies treatment; Sato etc., 2006, J Immunol176:7686-94).

Yoke closes in the mankind IFN-of 20-kDa Polyethylene Glycol λ 1 (PEG-IFN-λ 1) and is at present in the clinical development of chronic HCV infection treatment.In studying in the Ib stage, under each dosage (0.5-3.0 μ g/kg), observe antiviral activity, and when PEG-IFN-λ 1 being applied to the gene 1 type HCV patient of recurring after IFN-α therapy, virus load is down to 4.0log (Muir etc. from 2.3,2010, Hepatology52:822-32; Ramos, 2010, J Interferon Cytokine Res30:591-95).The IIb stage is studied (Zeuzem etc., 2011, J Hepatology54:5538-39) reported that HCV patient's (gene 1 type and 4 types) is for having significantly higher response rate with PEG-IFN-λ 1 treatment than the treatment of PEG-Intederon Alpha-2a, and utilize PEG-IFN-λ 1 than other advantages of utilizing PEG-Intederon Alpha-2a comprise that adverse events frequency is lower, the frequency of influenza-like symptom, anemia and musculoskeletal symptom reduces, and seldom observes neutrophilic granulocyte and reduce and thrombocytopenia.Yet, compare the visible more liver toxicity of height ratio (Zeuzem etc., 2011, J Hepatology54:5538-39) in maximum dose level PEG-IFN-λ 1 with PEG-Intederon Alpha-2a.In order to improve effect and the safety overview of IFN-λ 1, we have built module DOCK-AND-LOCK ^tM(DNL ^tM) complex ties this cytokine and be the targeting antibodies of the stabilisation dimeric forms of Fab albumen.

To be derived from the Fab-DDD2 module dimerization of humanized antibody hRS7 (anti-Trop2), hMN15 (anti-CEACAM6), hL243 (anti-HLA-DR) or c225 (inosculating antibody EGFT) and be connected to produce respectively immune cell factor (E1)-λ 1, (15)-λ 1, (C2)-λ 1 and (c225)-λ 1 with AD2-IFN-λ 1.Persons of ordinary skill in the art will recognize that the present invention is not limited to exemplary antibodies, but can utilize any known antibodies to implement.Evaluate the biological activity of interferon-antibody construct and compare with the recombinant human IFN-λ 1 (rhIFN-λ 1) in targeted cells system of institute and non-targeted cell line.The hRS7Fab dimer that we observe in (E1)-λ 1 makes IFN-λ 1 enlarge markedly (approximately 1000 times) for the vitro efficacy of several Trop2-positive cell line, and described cell line comprises human cervical cancer ME-180 (EC ₅₀< 0.1pM), lung squamous cancer SK-MES-1 (maximum 60% suppresses) and esophageal carcinoma TE-11 (maximum 45% suppresses).Similarly, at the positive ME-180 cell of CEACAM6-(EC ₅₀< 1pM) and in TE-11 cell, hMN15Fab dimer makes the growth inhibited of IFN-λ 1 mediation significantly strengthen (approximately 100 times), but really not so in the negative SK-MES-1 cell of CEACAM6.

In order to study (Fab) ₂the antiviral activity of-IFN-λ 1, we have compared rhIFN-λ 1, (15)-λ 1 and (C2)-λ 1 infects for encephalomyocarditis virus (EMCV) and in the impact of expressing CEACAM6 but not copying in the A549 cell of HLA-DR.Result shows, rhIFN-λ 1 and (Fab) ₂-IFN-λ 1 can prevent the cytopathic effect of EMCV induction effectively, but that (15)-λ 1 shows is higher 6 times and than the antiviral efficacy of high 10 times of (C2)-λ 1 than rhIFN-λ 1.Other studies show that, in the cell of institute's targeting, (Fab) ₂-IFN-λ 1 can enhancing signal transductant and the phosphorylation (critical events in the activation of Jak-STAT signal transduction) of transcription activator (STAT) 1,2 and 3, and the cell surface expression of I class ajor histocompatibility complex (MHC I), it promotes that antigen presents.These data show jointly, and the cell surface immobilization of the IFN-λ 1 mediating in conjunction with Fab antibody fragment by targeting can significantly strengthen its effect in the cell of institute's targeting and improve safety overview in body.Those skilled in the art will recognize that, these effects are not limited to Fab antibody fragment, but also there will be in complete antibody or other antibody fragments.These results show, comprise and be connected to the targeting antibodies of IFN-λ 1 or the DNL of antibody fragment ^tMcomplex can be used as therapeutic agent and treat cancer, infectious disease, asthma or multiple sclerosis.

Materials and methods

antibody and reagent-humanized antibody hA20-IgG (anti-CD20), hRS7-IgG (anti-Trop-2), hMN15-IgG (anti-CEACAM6) and hL243 (anti-HLA-DR) are from Immunomedics, Inc.Recombinant human IFN-λ 1, for the mice mAb of mankind IFN-λ 1 be purchased from R & D Systems Inc for the mice FITC-IgG1k of human HLA-ABC.Comprise be connected to the AD2 part of mutant (C171S) or wild type IFN λ 1 and the fusion rotein of poly-His sequence aminoacid sequence in demonstration below.Rabbit antibody for STAT1, STAT3, pY-STAT1 and pY-STAT3 is purchased from Cell Signaling Technology Inc.STAT2 antibody is from Santa Cruz Biotechnology.PY-STAT2 antibody is from Millipore Corporation.

Wild type: AD2-IFN-λ 1

MCGQIEYLAKQIVDNAIQQAGCEFPKPSTPPGSSGGAPAMDGPVPTS KPTTTGKGCHIGR FKSLSPQELASFKKARDALEESLKLKNWSCSSPVFPGNWDLRLLQVRERPVALEAELAL TLKVLEAAAGPALEDVLDQPLHTLHHILSQLQACIQPQPTAGPRPRGRLHHWLHRLQEA PKKESAGCLEASVTFNLFRLLTRDLKYVADGNLCLRTSTHPESTVEHHHHHH(SEQ?ID?NQ：96)

Mutant: AD2-IFN-λ 1-C171S

MCGQIEYLAKQIVDNAIQQAGCEFPKPSTPPGSSGGAPAMDGPVPTS KPTTTGKGCHIGR FKSLSPQELASFKKARDALEESLKLKNWSCSSPVFPGNWDLRLLQVRERPVALEAELAL TLKVLEAAAGPALEDVLDQPLHTLHHILSQLQACIQPQPTAGPRPRGRLHHWLHRLQEA PKKESAGCLEASVTFNLFRLLTRDLKYVADGNLSLRTSTHPESTVEHHHHHH(SEQ?ID?NO：97)

the expression of AD2-IFN-λ 1 and purification-in order to produce for DNL ^tMiFN-λ 1 module that complex forms, syntheticly comprises AD2, knuckle joint and for the synthetic DNA sequence (Fig. 8 A) of the mankind INF-λ 1 (C171S) that optimizes at expression in escherichia coli and be cloned in the Msc I and Xho I site of pET-26b carrier.With the Rosetta-pLysS cell (Novagen) that plasmid transforms, at 37 ℃, be supplemented with in the Difco 2xYT meat soup (Becton Dickinson) of 100 μ g/ml kanamycin sulfate and 34 μ g/ml chloromycetin and grow.When cell density reaches OD ₆₀₀be 1.0 o'clock, add IPTG to 0.5mM and at 30 ℃ induced protein express to continue 4 hours.By centrifugal collecting cell and at-80 ℃ freeze overnight.Agglomerate is thawed and dissolving buffer (2%Triton-X100,5mM MgSO ₄, 10 units per ml nucleases (benzonase) (Novagen), 100 μ M AEBSF, 20mM Tris-HCl, pH8.0) in homogenize.Homogenate under 18,000RPM centrifugal 30 minutes and agglomerate is again homogenized in PBS/1%Triton X-100, and then form agglomerate.Agglomerate is dissolved in the 6M guanidine hydrochloride, 100mM sodium phosphate (pH8.0) of 20ml and to be loaded to His-Select affinity column (GE-Healthcare) upper, then uses 6M guanidine, 50mM sodium phosphate (pH8.0) washing.At 4M guanidine hydrochloride, 100mM NaH ₂pO ₄(pH4.5) elute protein in, and by adding 3M Tris-HCl (pH8.6) neutralization of 200 μ L, add DTE to 60mM and make solution at room temperature keep spending the night.The denatured protein solution rapid dilution of reduction, in 0.5M arginine, 20mM oxidized form of glutathione, 2mM EDTA, 100mM Tris (pH8.0), is then dialysed and at 4 ℃, kept 72 hours for 5L renaturation buffer (0.5M arginine, 2mM oxidized form of glutathione, 0.6mM DTE, 2mM EDTA, 20mM Tris (pH8.0)).Then for PBS-AG-2 buffer (35.2mM Na ₂pO ₄.7H ₂o; 0.4M NaCl; 6.5mM NaH ₂pO ₄.H ₂o; 150mM arginine; 150mM glutamic acid, pH8.0) dialysis solution.End product is concentrated into about 0.5mg/ml and utilizes SDS-PAGE to analyze.

dNL ^tM construct-by hRS7-, hMN15-or hL243-Fab-DDD2 module and AD2-IFN-λ 1 module fusion formation DNL ^tMcomplex generates (Fab) ₂-IFN-λ 1 conjugates, comprises (E1)-λ 1, (15)-λ 1 and (C2)-λ 1 (Fig. 8 B), as discussed previously (referring to for example U.S. Patent No. 7,521,056,7,527,787,7,550,143,7,534,866,7,666,400,7,858,070,7,871,622,7,901,680,7,906,118,7,906,121,7,981,398 and 8,003,111, its embodiment separately is partly incorporated herein by reference).According to (the accession number: DB00002) build c225-Fab-DDD2 module of the sequence from DrugBank.Sequentially on Kappa-select and His-select post purified product and utilize SDS-PAGE reduction and non-reduced condition under use 4-20%Tris-glycine gels (not shown) to analyze.

cell culture and surface combination-TE-11 is provided by Hiroshi doctor Nakagawa (University of Pennsylvania) friendship.Huh-7 and T.Tn are purchased from Japanology living resources preservation center (Japanese Collection of Research Bioresources).Every other cell line is purchased from American type culture collection.ME-180, TE-11 and A375 cell be growth in containing the RPMI1640 culture medium (Invitrogen) of 10%FBS (Hyclone).HepG2, Huh-7 and SK-MES-1 cell be growth in containing the EMEM culture medium (ATCC) of 10%FBS.A549 is growth in containing the F12 culture medium (InVitrogen) of 10%FBS, and T.Tn growth in containing the DMEM/F12 culture medium (InVitrogen) of 10%FBS.All cells system all at 37 ℃ at 5%CO ₂humid atmosphere in cultivate.

For binding assay, cell is carried out to of short duration trypsinization, be suspended in fresh culture and form agglomerate, then with 10 μ g/ml humanization mAb or in 1%BSA-PBS the agent based on IFN-λ 1 of serial dilution again suspend.At 4 ℃, incubation is after 45 minutes, cell is formed to agglomerate and uses 1%BSA-PBS washed twice, at 4 ℃ together with the anti-human class IgG-Fc of goat of FITC labelling or mouse anti human class IFN-λ 1 incubation 45 minutes, then with goat anti-mouse IgG-Fc of FITC labelling, survey.After three washings, utilize fluidic cell surveying to measure combination.The variation of expressing in order to study MHC I, makes cell be exposed to 1 dose of IFN-λ and continues 3 days, and be combined to detect its surperficial MHC1 by the mouse IgG 1k of the FITC labelling with for human HLA-ABC.Use the non-specific mouse IgG 1k of FITC labelling as negative control.

in-vitro multiplication-ME-180, SK-MES-1, TE-11 and T.Tn cell are seeded in 96 orifice plates and at 37 ℃ and are incubated overnight with the density of 1000 cells/well, be then exposed to 1 dose of the IFN-λ of progressive concentration, continue 4 days.Use CellTiter96 cell proliferating determining method (Promega) to measure viable cell density.

antiviral algoscopy-use the stable Huh-7 cell line (being called Huh-7-Con1) that contains HCV genotype 1 b Con1 replicon, utilize HD Biosciences (China) Co., Ltd (Shanghai, China) measures the anti-HCV activity of IFN-λ 1 and 2 (Fab)-λ 1.Firefly luciferase gene is incorporated in this replicon to the reporter gene as virus levels.This algoscopy comprises 1 dose of three kinds of IFN-λ, (c225)-λ 1, (C2)-λ 1 and rhIFN-λ 1.Wherein, two Fab of the chimeric mAb that (c225)-λ 1 comprises the EGFR on selectively targeted Huh-7 cell surface, and (C2)-λ 1 and rhIFN-λ 1 are two non-targeted testers.Huh-7-Con1 cell is processed 3 days with these three kinds of agent, and measures virus replication level by measuring uciferase activity.Meanwhile, also use CellTiter Glo test kit (Promega) to evaluate the cytotoxicity of these agent for parent Huh-7 cell.

In another algoscopy, use and utilize the cytopathic effect that PBL Interferon Source (Piscataway, NJ) carries out to suppress algoscopy at the antiviral activity with measurement (15)-λ 1 on the A549 cell of EMCV.This algoscopy comprises that hMN-15-Fab-DDD2 is as negative control, and rhIFN-λ 1 reference material (PBL Interferon Source) is as positive control, and (C2)-λ 1 is as the non-targeted contrast of structural correspondence thing.

immunoblotting (Western blot)-by ME-180, HepG2 and A375 cell with 5 * 10 ⁵the density of individual cells/well is coated with to be laid in 6 orifice plates and at 37 ℃ and is incubated overnight.In order to evaluate STAT activation, with 1 dose of IFN-λ shown in the concentration of successively decreasing, process cell 1 hour, then use PhosphoSafe ^tMextracting reagent (EMD) dissolves.The cell lysates of emanating on SDS-PAGE, be transferred on NC Nitroncellulose film (Bio-Rad), then use rabbit antibody and phosphotyrosine specific antibody pY-STAT1, pY-STAT2 or pY-STAT3 generation trace for total STAT1, STAT2 or STAT3, by HRP-goat anti-rabbit antibodies, detect.Beta-actin antibody is contrasted as loading.

To (E1)-λ 1 of female athymic nude mice (Taconic, Germantown, NY) the subcutaneous injection 2.4nmol in four groups of 8 week ages and 6 hours, 16 hours, 24 hours and blood sampling in 48 hours.Use enzyme-linked immunosorbent assay (ELISA) to measure the serum-concentration of complete (E1)-λ 1.Use the non-compartment analysis program of WinNonLin (5.3 editions, Pharsight Corporation, St.Louis, MO) to calculate pharmacokinetic parameter.In order to confirm the result of ELISA, also by the vitro proliferation algoscopy of ME-180 cell, use (E1)-λ 1 as reference material, evaluated the biological activity of IFN-λ 1 in selectivity blood serum sample.

rT-PCR analyzes.with 1 dose of IFN-λ, process HepG2 cell 24 hours and use the separated total RNA of reagent (Life Technologies).Use iII single step RT-PCR system (Life Technologies) utilizes forward and reverse primer analyze under the following conditions the mrna expression of myxovirus resistance A (MxA) gene: a cDNA lasting circulation in synthetic-55 ℃/30 minutes and within PCR-94 ℃/15 seconds, 62 ℃/30 seconds, 68 ℃/30 seconds, continue 25 circulations.With the similar condition of internal contrast under the increase 452-bp fragment of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA.

pharmacokinetics in mice.to (E1)-λ 1 of female athymic nude mice (Taconic, Germantown, NY) the subcutaneous injection 2.4nmol in four groups of 8 week ages and 6 hours, 16 hours, 24 hours and blood sampling in 48 hours.Use enzyme-linked immunosorbent assay (ELISA) to measure the serum-concentration of complete (E1)-λ 1.Use the non-compartment analysis program of WinNonLin (5.3 editions, Pharsight Corporation, St.Louis, MO) to calculate pharmacokinetic parameter.In order to confirm the result of ELISA, also by the vitro proliferation algoscopy of ME-180 cell, use (E1)-λ 1 as reference material, evaluated the biological activity of IFN-λ 1 in selectivity blood serum sample.

statistical analysis.use Prism GraphPad software kit (Advanced Graphics Software, Rancho Santa Fe, CA) to measure resultful statistical significance (P < 0.05) with F check.

Result

(Fab) ₂ the generation of-IFN-λ 1 and sign-point mutation (C171S) is introduced in wild type IFN-λ 1 sequence to the potential interference that does not match cysteine residues that there is the refolding of DNL module and assembling to eliminate.In escherichia coli, produce AD2-IFN-λ 1 module recombinant forms and by immobilized metal affinity chromatography (IMAC) under Denaturing from inclusion body purification.With dithiothreitol, DTT (DTE), go back crude protein, then in the refolding buffer that contains oxidized form of glutathione, refolding forms disulfide bond with permission.As utilizing SDS-PAGE (not shown) to be measured, AD2-IFN-λ 1 protein is by highly purified and mainly exist with free state.The output of end product is about 6mg/ml Bacillus coli cells culture.

As shown in Fig. 8 B, by AD2-IFN-λ 1 module and Fab-DDD2 module are combined to generate (Fab) ₂-IFN-λ 1 conjugates.In current research, the Fab-DDD2 module of hRS7, hMN15 or hL243 is mixed with the AD2-IFN-λ 1 of molar excess, and be incubated overnight with the reduced glutathion of 1mmol/L, add subsequently oxidized form of glutathione (2mmol/L).Utilize Kappa-select column purification reactant mixture, and successfully generate four kinds of conjugatess (E1)-λ 1, (15)-λ 1, (C2)-λ 1 and (c225)-λ 1.Utilize SDS-PAGE to show the purity of these conjugatess, its three bands of a spectrum of emanating (Fab DDD2-heavy chain, Fab light chain and AD2-IFN-λ 1) and the main high molecular band (not shown) of emanating in reproducibility gel in irreducibility gel.

the cell surface expression of antigen-by fluidic cell surveying, measure Trop-2, CEACAM6, HLA-DR and EGFR at seven kinds of human cancer cell lines (cervical cancer, ME-180; Esophageal carcinoma, TE-11; Pulmonary carcinoma, A549, SK-MES-1; Hepatocarcinoma, HepG2, Huh-7; And melanoma-skin carcinoma, the expression on cell surface A375).As shown in Figure 9, Trop-2 highly expresses on ME-180 and TE-11 cell, and moderate is expressed on SK-MES-1 cell.Except A375 and SK-MES-1, CEACAM6 expresses in every other cell line, and wherein the level on A549 and HelpG2 cell is the highest.A375 cell line shows the high expressed of HLA-DR.Observe EGFR for the high expressed of Huh-7, ME-180, TE-11, SK-MES-1 and A549.These data provide (Fab) ₂the basis of the further research of the cell-specific targeting of-IFN-λ 1.

(Fab) ₂ the immobilization of the enhancing of-IFN-λ 1 on cell surface-compare with AD2-IFN-λ 1 module, (Fab) ₂-IFN-λ 1 shows and the binding affinity of institute's targeted cells significantly strengthens.Under the concentration of 8nM, as detected by mouse anti human class IFN-λ 1mAb, than AD2-IFN-λ 1 module, (E1)-λ 1 is for the binding signal high 107 times (Figure 10 A) of ME-180, (15)-λ 1 is for the binding signal high 15 times (Figure 10 B) of HepG2, and (C2)-λ 1 is for the binding signal high 508 times (Figure 10 C) of A375 cell.These data show with (Fab) ₂the IFN-λ 1 that yoke closes is presented in the immobilization significantly strengthening on the cell surface of institute's targeting.

growth in vitro suppresses and cytotoxicity-identify that cervical cancer ME-180 is to the extremely sensitive cell line of IFN-λ 1.RhIFN-λ 1 complete cell growth inhibiting almost under the concentration higher than 10nM, and AD ₂-IFN-λ 1 module shows the activity (EC equating with rhIFN-λ 1 ₅₀=0.1nM, data are not shown).In targetable conjugate compound (E1)-λ 1, IFN-λ 1 significantly strengthens (EC for the activity specific of ME-180 ₅₀< 0.1pM), its combination than AD2-IFN-λ 1 or AD2-IFN-λ 1 and AD2-hRS7-Fab high 1000 times above (Figure 11).Compare with ME-180, lung squamous cancer SK-MES-1 and esophageal carcinoma TE-11 cell are less and only show the IFN-of maximum concentration λ 1 time the growth inhibited (not shown) that is respectively maximum 60% and 45% to the sensitivity of IFN-λ 1.Yet, in these two kinds of cell lines, still observe the activity of the enhancing of (E1)-λ 1, the high approximately 1000 times of (not shown) of combination than AD2-IFN-λ 1 or AD2-IFN-λ 1 with AD2-hRS7-Fab.In addition, we have evaluated the activity of (15)-λ 1 for the positive ME-180 of CEACAM6-and TE-11 cell line.As shown in Figure 12, (15)-λ 1 represents than the activity of high 100 times of the combination of AD2-IFN-λ 1 or AD2-IFN-λ 1 and hM[N15-IgG-AD2.(15)-λ 1 is for the EC of ME-180 ₅₀for about 1pM, it is higher 10 times than (E1)-λ 1.

antiviral activity-in Huh-7 and A549 cell, measured respectively the antiviral activity of selectivity 2 (Fab)-λ 1 construct for HCV and EMCV.In expressing the Huh-7 cell of EGFR, when inhibition HCV copies, (c225)-λ 1 (EC ₅₀=0.56pM) than non-targeted property (C2)-λ 1 (EC ₅₀=91.2pM) and business rhIFN-λ 1 (EC ₅₀=69.2pM) respectively more effective 163 times and 123 times (Figure 13 A).In expressing the A549 cell of CEACAM6, (15)-λ 1 shows respectively the anti-EMCV activity (Figure 13 B) of high 10 times and 6 times than non-targeted property (C2)-λ 1 and rhIFN-λ 1.It should be noted that (C2)-λ 1 has retained 65% to 75% antiviral activity of rhIFN-λ 1.The cell surface targeting of these result indications IFN-λ 1 can effectively strengthen its antiviral activity.

cellular signal transduction-in order to understand (Fab) ₂the mechanism that strengthens IFN-λ 1 activity, we have compared (Fab) ₂-IFN-λ 1 and AD2-IFN-λ 1 signaling activity in three kinds of cell lines.Phosphorylation assay Faxian shows that 15-Fab-DDD2 does not induce any STAT phosphorylation in HepG2 cell, and AD2-IFN-λ 1 induces detectable phosphorylation (not shown) under 0.1nM concentration.By contrast, the significantly larger phosphorylation of (15)-λ 1 induction three kinds of STAT, particularly STAT1, demonstrates the signaling activity of 0.01nM (15)-λ 1 no better than 0.1nMAD2-IFN-λ 1 (not shown).In the A375 cell of the ME-180 of (E1)-λ 1 targeting and (C2)-λ 1 targeting, also shown (Fab) ₂-IFN-λ 1 strengthens (data are not shown) with respect to the STAT phosphorylation of AD2-IFN-λ 1.

Also by fluidic cell surveying, studied the ability (not shown) that (15)-λ 1 raises the cell surface expression of MHCI class antigen (MHC-I) in HepG2 cell.And process with the hMN-15-Fab-DDD2 of 1nM at the most the surface level unchanged (MFI approximately 50) that HepG2 cell continues three angel MHC-1, in the cell of processing at (the 15)-λ 1 that is low to moderate 1pM, observing MFI increases more than 3 times (approximately 170).(15)-λ of 100pM 1 time, the expression of MHC-1 reaches maximum horizontal (MFI approximately 270).Although AD2-IFN-λ 1 can raise MHC-1, it needs just effective (not shown) of higher concentration (100pM).

We have also studied the ability of the expression of (15)-λ 1 induction myxovirus resistance A (MxA) gene (the bioactive reliable label of IFN), and utilize RT-PCR measurement result.In HepG2 cell untreated or that processed by hMN-15-Fab-DDD2, utilize RT-PCR to fail to detect the mRNA (not shown) of MxA.In by the cell of 0.1pM (15)-λ 1 or 10pM AD2-IFN-λ 1 processing, the inducing action of MxA gene is obvious (not shown), has therefore further proved that (15)-λ 1 is better than AD2-IFN-λ 1, particularly general 2 (Fab)-λ 1 and is better than the advantage of IFN-λ 1.

pharmacokinetics in mice.after the subcutaneous administration of single dose (2.4nmol), the average serum concentration of complete (E1)-λ 1 reached high level and in the time of 48 hours, is down to the following (not shown) of ELISA detection limit in the time of 6 hours.Pharmacokinetics (PK) the parameter display mean residence time that is derived from non-compartment analysis is 12 hours, T _1/2be 8.6 hours, and clearance rate is 2.2ml/h.When using MTS algoscopy also to measure its concentration in blood serum sample by (E1)-λ 1 for the inhibition activity of ME-180 cell, result is consistent (not shown) to a great extent.

Restructuring IFN-α has clearance rate very fast in mice, shows that mean residence time is only 0.7 hour.Therefore, 2 (Fab)-λ 1 show the PK (not shown) of the remarkable improvement suitable with PEG-IFN-λ with PEG-IFN-α [37].

Discuss

The type iii interferon (IFN) that comprises IFN-λ 1, IFN-λ 2 and IFN-λ 3 cause aspect antiviral, antitumor and immunoregulatory activity similar with IFN-α behavior.Due to its more limited cell target, IFN-λ is attractive for the potential substitute of the existing therapeutic scheme as based on IFN-α.By by the stabilisation Fab dimer that is derived from hRS7 (the anti-Trop-2 of humanization), hMN-15 (the anti-CEACAM6 of humanization), hL243 (the anti-HLA-DR of humanization) and c225 (inosculating antibody EGFR) and IFN-λ 1 locus specificity be tied in together with, form the Novel immune cytokine that is called as respectively (E1)-λ 1, (15)-λ 1, (C2)-λ 1 and (c225)-λ 1, we have prepared and have comprised the DOCK-AND-LOCK that antibody yoke closes IFN-λ 1 ^tMcomplex is to improve the antiproliferative effect (1,000 times at the most) in IFN-λ 1 system of the cancer cell at institute's targeting.Compare with non-targeted property (C2)-λ 1, by (15)-λ 1 or (c225)-λ 1 IFN-λ 1 targeted delivery to antigen presentation cell separately has also significantly been increased to antiviral activity, as (the EC in mankind's adenocarcinoma of lung epithelial cell line A549 of (the 15)-λ 1 for encephalomyocarditis virus ₅₀=22.2pM is to 223pM) and for (c225)-λ 1 (EC in Polymerase chain reaction Huh-7 of hepatitis C virus ₅₀=0.56pM is to 91.2pM) shown in.These astonishing and unpredictable consequences are better location and the stronger combinations to the cell of antibody institute targeting owing to IFN-λ 1, and the favourable pharmacokinetic profile (T of (E1)-λ 1 in mice _1/2=8.6 hours).

Trop-2 and CEACAM6 are with the higher horizontal expression (not shown) on target cell of the receptor than IFN-λ 1.Therefore, IFN-λ 1 molecule more than 10 times can DNL conjugates form be incorporated into target cell.Also can make being connected altogether of the allos dimerization receptor of Trop-2 or CEACAM6 and IFN-λ 1 almost 100 times of the bond strength increases of IFN-λ 1, if (E1)-λ 1 is as shown in ME-180.The targeted delivery of the IFN-λ 1 that immunity yoke closes provides the significantly larger biological activity for tumor and infectious disease treatment than the antibody of using alone or in combination respectively and interferon.

Utilizing PEG-IFN-λ 1 to explore IFN-λ as the potentiality of the treatment substitute of IFN-α, it has shown the safety overview (Miller etc. that increase than PEG-IFN-α-2a in clinical research, 2009, Ann N Y Acad Sci1182:80-87; Ramos, 2010, J Interferon Cytokine Res30:591-95).Yet, for the patient who processes with PEG-IFN-λ 1 and PEG-IFN-α-2a, the incidence rate of some serious adverse events (comprising dose limitation liver toxicity) is similar, and even more frequent (Zeuzem etc. in the patient who processes at the PEG-IFN-λ 1 with maximum dose level, 2011, J Hepatology54:5538-38).On the other hand, for some cancer (Meager etc., 2005, Cytokine31:109-18) or virus (Ank etc., 2006, J Interferon Cytokine Res26:373-79), IFN-λ not as I type IFN effective.The specific antibody of surface antigen tool that our supposition is connected to IFN-λ for great expression can strengthen it in the location of target cell, thereby produces larger effect and be hopeful to make the toxicity for non-target cell less.Therefore, we have developed the prototype of four kinds of immune cell factors based on IFN-λ, and each self-contained locus specificity yoke closes the dimeric IFN-λ 1 of stabilisation in Fab, and have shown that it closes the superiority of parent's module than not yoke alone or in combination.In antitumor and antiviral algoscopy, shown the effect improving, and this is consistent with cell surface combination and the signal transduction of the enhancing that can be realized by composition antibody.

In antitumor research, we have determined that ME-180 is as the cell line the most responsive to IFN-λ 1, and it has more than 80% maximum inhibitory action and EC ₅₀than other cell lines low 20 times of (Zitzman etc., 2006, the BBRC344:1334-41 that report in document; Meager etc., 2005, Cytokine31:109-18; Maher etc., 2-8, Cancer Biol Ther7:1109-15; Guenterberg etc., 2010, Mol Cancer Ther9:510-20).In addition, the great expression of Trop-2 on ME-180 makes these tumor cells extremely sensitive to (E1)-λ 1, under 1fM, growth inhibited effect and EC can be detected ₅₀(< 0.1pM) is than AD2-IFN-λ 1 (EC ₅₀about 100pM) low 1000 times.In TE-11, SK-MES-1 and other cancer cell system, also observe the similar enhancing that suppresses effect.Because IFN-λ also induces congenital and adaptive immunity reaction, this evaluates herein, and consider the recent research that shows the constructive expression of IFN-λ in the several muroid cancer cell that comprises B16 melanoma, BNL hepatoma and MCA205 fibrosarcoma is, although antiproliferative activity in shortage body, but the growth and metastasis of tumours (Numasaki etc. in homogenic mouse model have significantly been suppressed by raising immunocyte and relevant cell factor, 2007, J Immunol178:5086-98; Abushahba etc., 2010, Cancer Immunol Immunother59:1059-71:Lasfar etc., 2006, Cancer Res66:4468-77), we suppose that the targeted delivery of IFN-λ will be similar to the constructive expression of IFN-λ in cancerous cell, thus the cytotoxicity (Pardoll and the Drake that in immunotherapy for cancer, produce local immunity reaction and strengthen, 2012, J Exp Med209:201-9).

In antiviral study, (c225)-λ 1 and the HCV genotype 1 b Con1 replicon of take are that main the selectively targeted of the positive Huh-7 cell of EGFR-shown that antiviral efficacy strengthens respectively 123 times and 163 times than non-targeted property rhIFN-λ 1 and (C2)-λ 1.In another algoscopy, the positive A549 cell of CEACAM6-that (15)-λ 1 targeting is excited with EMCV has shown that antiviral protection improves respectively 6 times and 10 times than non-targeted property rhIFN-λ 1 and (C2)-λ 1.(c225)-λ 1 and effect difference between (15)-λ 1 may be that the cell/viral system due to its targeting is different to the sensitivity of IFN-λ 1.In previous research (Marcello etc., 2006, Gastroenterol131:1887-98), in Huh-7/HCV system, rhIFN-λ 1 is lower 10 times than rhIFN-α effect, but in A549EMCV effect low 210 times (Meager etc., 2005, Cytokine31:109-18).Although the antiviral activity of (15)-λ 1 induction strengthens not as (c225)-λ 1 height in target cell, but consider that Pegylation IFN only retains the not approximately activity below 30% of Pegylation IFN, this remains the significant (Grace etc. of discovery, 2005, J Biol Chem280:6327-36), and the activity specific of AD2-IFN-λ 1 and rhIFN-λ 1 similar.Therefore, 2 (Fab)-λ 1 can allow than the dosage regimen that dosage is lower and frequency is identical or less of PEG-IFN-λ 1 used in current clinical research.

As therapeutic agent, restructuring IFN is subject to its restriction of clearance rate very fast.Research based on previous, restructuring IFN-α-2b, PEG-IFN-α-2a and PEG-IFN-α-2b show respectively 0.7 hour, 14.9 hours and 9.3 hours half-life (Rossi etc., 2009, Blood114:3864-71).Therefore it is likely that, (E1)-λ 1 half-life (8.6 hour) suitable with PEG-IFN-α in mice used for interior therapeutic.

Embodiment 10. is used for the treatment of Alzheimer (Fab) ₂the DNL construct of-interferon-λ 1

Alzheimer (AD) is that a kind of Clinical symptoms is carrying out property hypomnesis, confusion, health runs down and final dead degenerative brain disease.1,500 ten thousand people that have an appointment in worldwide are subject to Alzheimer and invade and harass.On histology, this disease be characterized as neuritic plaques, be mainly present in association's cortex, limbic system and ganglion basal.The main component of these specklees is amyloid beta peptide (A β), and it is the pyrolysis product of amyloid beta-protein precursor (β APP or APP).

A β seems to have pivotal role in the neuro pathology of Alzheimer.Family's form of this disease (Tanzi etc., 1996, Neurobiol.Dis.3:159-168 relevant to senilism gene the sudden change of APP; Hardy, 1996, Ann.Med.28:255-258).Disease association sudden change in these genes causes the generation of the A β of 42 amino acid form to increase, and described form is the principal mode existing in amyloid speckle.With mankind A β, make the transgenic mice immunity of the APP of overexpression disease association mutant forms can reduce speckle burden and relevant diseases (Schenk etc., 1999, Nature400:173-177; WO99/27944).For the periphery of the antibody of A β, use speckle burden (Bard etc., 2000, the Nature Medicine6 (8): 916-919 also reducing in brain; WO2004/032868; WO00/72880).

The treatment that antibody therapy is Alzheimer and prevention provide a kind of method likely.Yet the human clinical trial who carries out with the vaccine that comprises A β 1-42 is because the meningoencephalitis in patient's subset suspends.Orgogozo etc., Neruology61:7-8 (2003); Ferrer etc., Brain Pathol.14:11-20 (2004).With N terminal specific anti-amyloid beta antibodies, carry out passive immunity and cause diffusivity amyloid significantly to reduce, but the micro-hemorrhage frequency of the brain of transgenic mice increases.Pfeifer etc., Science298:1379 (2002).Still need to have antibody and/or the immune conjugate of the improvement that is used for the treatment of Alzheimer of the toxicity of reduction.

According to embodiment 9, prepared the DNL that comprises the alemtuzumab that is connected to interferon-λ 1 ^tMcomplex.The human patients intravenous of suffering from Alzheimer to diagnosis with the dosage of 2mg is used interferon-antibody complex, and weekly twice, continue 4,8 or 12 weeks.By neuropsychology thermometrically effect, described test comprises ADAS-cog and the complete neuropsychology test of CERAD.

In all patients except lowest dose level processed group, slight (15%) of after the processing of 12 weeks, observing ADAS-cog improves.For simple and easy mental status examination (MMSE), observe similar result of study.In 4/10ths patient, vision structuring capacity improves.Do not observe the serious ill effect that interferon-antibody is used.

According to embodiment 9, prepared to comprise and be connected to the meter La Zhu monoclonal antibody (anti-CD74 humanization IgG) of interferon-λ 1 or the DNL of its Fab fragment ^tMcomplex.With the dosage of twice 2mg weekly, to 75 years old women's intravenous suffering from early stage Alzheimer, use this complex, continue 8 weeks.After each transfusion, notice some slight feeling sick and hypotension sign, this disappeared in 3 days subsequently.By Cognitive Aptitude Test (ADAS-cog) and the complete neuropsychology of CERAD test, when baseline and after treatment, measure effect when 2 weeks and 8 weeks.Result indication, cognitive competence and general psychology and exchange status improve 20%.After 4 months, repeat this treatment, the well tolerable property of its tool, and patient shows maintaining that cognitive competence improves, and comprises vision structuring capacity.

According to embodiment 9, prepared to comprise and be connected to the hL243 (anti-HLA-DR humanization IgG) of interferon-λ 1 or the DNL of its Fab fragment ^tMcomplex.With the dosage of twice 1mg weekly, to 82 years old women's intravenous suffering from Alzheimer, use this complex, continue 8 weeks.After each transfusion, notice some slight feeling sick and hypotension sign, this disappeared in 3 days subsequently.By Cognitive Aptitude Test (ADAS-cog) and the complete neuropsychology of CERAD test, when baseline and after treatment, measure effect when 2 weeks and 8 weeks.Result indication, cognitive competence and general psychology and exchange status improve 17%.After 4 months, repeat this treatment, the well tolerable property of its tool, and patient shows maintaining that cognitive competence improves, and comprises vision structuring capacity.

Embodiment 11. is used for the treatment of asthma (Fab) ₂the DNL construct of-interferon-λ 1

Asthma is a kind of heterogeneous familial disease, it is characterized in that tracheal bronchus is for the high response stimulating.Clinically, asthma shows as dense sticky stagnant, the dyspnea of tracheobronchial extensively narrow, secretions, coughs and pant, thereby causes the abnormal distribution of airway resistance increase, pulmonary and chest excessive expansion, ventilation and lung blood flow.This disease shows as between asymptomatic stage to be interted the acute paresthesia epilepsy phase.Acute attack causes anoxia, and may be fatal.Approximately 3% of total world population suffers from this disease.Symptoms of asthma may be aggravated by triggering the existence of antigen and level, environmental factors, occupational factor, physical demands and emotional stress.Although asthma can for example, for example, for example, for example, be treated with methylxanthine (theophylline), beta-adrenergic agonist (catecholamine, resorcinol, bigcatkin willow aglycon and ephedrine), glucocorticoid (hydrocortisone), mastocyte threshing inhibitor (being that chromone class is as sodium cromoglicate) and anticholinergic (atropine), but still need to be used for the treatment of the improved method and composition of asthma.

There is the acute attack of flu and its asthma in 80 years old male's Bronchial Asthmas, cough produces a large amount of dense thick yellow mucus frequently.Through every day 6 day time, by aerosol, suck to him and use (15)-λ 1 for twice.Dispersal risk-interferon compound as described in example 9 above.In several hours that suck for the first time, his breathing obviously improves, and coughs not frequent.When this therapeutic process finishes, his physical demands situation seems to be improved, and emotional state is also like this, and this is because his breathing has been improved and mucus cough significantly reduces.

Embodiment 12. is used for the treatment of multiple sclerosis (Fab) ₂the DNL construct of-interferon-λ 1

Multiple sclerosis (MS) is a kind of autoimmune disease, and wherein autoreactive T cell passes through blood brain barrier and attacks myelin, produces inflammation and causes demyelination and axonal degeneration.In person between twenty and fifty, after the uncertain outbreak of delayed ischemic neurological deficits, be that health and cognitive disorder accumulate gradually in discrete attack (recurrence type) or in time.Although also there is no known cure method, interferon-beta therapy is the current first-line treatment for recurrent remission form multiple sclerosis, and it shows effect aspect relapse rate reduction.Yet IFN therapy is associated with remarkable sickness rate, there is side effect such as the development of influenza-like symptom, bone marrow depression, autoimmune disease or deterioration, neutrophilic granulocyte minimizing, thrombocytopenia and neural spiritual influence.

Prepared as described in example 9 above the DNL that comprises the alemtuzumab that is connected to interferon-λ 1 ^tMcomplex.Every surrounding is used 2mg complex to the 64 years old women who suffers from recurrent multiple sclerosis (RMS).After the treatment of 12 months, in complex situation, observe the Risk Reduction that annual relapse rate decline and anergy continue progress.Compare with base line measurement result, notice that the gadolinium enhancing focus of each T1 weighted mri scanning significantly reduces.Side effect comprises 1/2 grade of infusion reaction, 1 grade of hypotension and some allergy, these all available corticosteroid well control.

Claims (37)

1. a method for the treatment of cancer, viral infection, asthma or multiple sclerosis, it comprises:

A) obtain interferon-antibody complex, it comprises:

I) the first fusion rotein, interferon-λ that it comprises anchoring structure territory (AD) part that is connected to AKAP protein;

Ii) the second fusion rotein, it comprises the dimerization that is connected to human kinase protein A (PKA) RI α, RI β, RII α or RII β and antibody or the antigen binding antibody fragment of stopping domain (DDD) part; And

B) to the experimenter with cancer, viral infection, asthma or multiple sclerosis, use described interferon-antibody complex;

Wherein the described DDD of two copies partly forms dimer, and described dimer is incorporated into described AD part to form described complex.

2. the method for claim 1, the group that wherein said interferon-λ selects free human interferon-λ 1, interferon-λ 2 and interferon-λ 3 to form.

3. the method for claim 1, wherein said antibody or antibody fragment are incorporated into the freely antigen of the following group forming of choosing: carbonic anhydrase IX, CCCL19, CCCL21, CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, AFP, PSMA, CEACAM5, CEACAM-6, B7, the ED-B of fibronectin, factor H, FHL-1, Flt-3, folacin receptor, gp41, gp120, GRO-β, HMGB-1, hypoxia inducible factor (HIF), HM1.24, insulin-like growth factor-i (ILGF-1), IFN-λ, IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, PAM4 antigen, NCA-95, NCA-90, Ia, HM1.24, EGP-1, EGP-2, HLA-DR, tenascin, Le (y), RANTES, T101, TAC, Tn antigen, Thomson-Friedenreich antigen, neoplasm necrosis antigen, TNF-α, TRAIL receptor (R1 and R2), VEGFR, EGFR, PlGF, complement factor C3, C3a, C3b, C5a, C5 and oncogene products.

4. the method for claim 1, wherein said antibody or its antibody fragment are incorporated into the freely antigen of the following group forming of choosing: TROP-2, CEACAM5, CEACAM6, HLA-DR, CD19, CD20, CD22, CD52, CD74 and EGFR.

5. the method for claim 1, the group that wherein said antibody or antibody fragment select free hLL1, hLL2, RFB4, hRS7, hPAM4, hMN-3, hMN-14, hMN-15, hMu-9, Immu31, hL243, hA19, hA20, alemtuzumab and hR1 to form.

6. the method for claim 1, the group that wherein said antibody fragment selects free Fab, Fv, scFv and dAb to form.

7. the method for claim 1, it further comprises to described experimenter uses other treatment agent.

8. method as claimed in claim 7, the group that wherein said therapeutic agent selects free second antibody, second antibody fragment, the second interferon, medicine, toxin, enzyme, hormone, immunomodulator, cytokine, chemotactic factor, antisense oligonucleotide, siRNA, RNAi, radionuclide, boron compound, photosensitizer, anti-angiogenic agent and short apoptosis agent to form.

9. method as claimed in claim 8, wherein said medicine choosing is the following group forming freely: 5-fluorouracil, APL, azaribine, Anastrozole, anthracycline, bendamustine, bleomycin, bortezomib, Bruton inhibitors of kinases, Bryostatin-1, busulfan, calicheamycin, camptothecine, carboplatin, 10-hydroxycamptothecine, carmustine, celecoxib, chlorambucil, cisplatin (CDDP), Cox-2 inhibitor, irinotecan (CPT-11), SN-38, carboplatin, carat Qu Bin, camptothecin, cyclophosphamide, cytosine arabinoside, dacarbazine, Docetaxel, dactinomycin, daunorubicin, amycin, 2-pyrrolinyl amycin (2P-DOX), cyano group-morpholinyl amycin, amycin glucosiduronic acid, epirubicin glucosiduronic acid, estramustine, table podophyllotoxin, estrogen receptor bonding agent, etoposide (VP16), etoposide glucosiduronic acid, etoposide phosphate, floxuridine (FUdR), 3 ', 5 '-O-dioleoyl-FudR, fludarabine, flutamide, farnesyl-protein transferase inhibitor, gemcitabine, hydroxyurea, idarubicin, ifosfamide, leunase, lenalidomide, formyl tetrahydrofolic acid, lomustine, chlormethine, melphalan, purinethol, Ismipur, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, nvelbine, nitroso ureas, plicamycin, procarbazine, paclitaxel, pentostatin, PSI-341, raloxifene, semustine, streptozotocin, tamoxifen, taxol, temozolomide, anti-platinum, Thalidomide, thioguanine, thio-tepa, teniposide, topotecan, tyrosine kinase inhibitor, uracil mustard, vinorelbine, vinblastine, vincristine, vinca alkaloids, LFM-A13, Dasatinib, imatinib and AMN107.

10. method as claimed in claim 8, the group that wherein said toxin selects free Ricin, abrin, alpha toxin, saporin, ribonuclease (RNA enzyme), ranpirnase, DNA enzyme I, staphylococcus enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, diphtherotoxin, Rhodopseudomonas extracellular toxin and Rhodopseudomonas endotoxin to form.

11. methods as claimed in claim 8, wherein said anti-angiogenic agent choosing is the following group forming freely: angiostatin, bar chalone, canstatin, mammary gland silk presses down albumen, VEGF antibody, anti-PlGF antibody, laminin，LN peptide, fibronectin, plasminogen activator inhibitor, tissue inhibitor of metalloproteinase, interferon, interleukin 12, IP-10, Gro-β, thrombospondin, 2ME2, proliferin associated protein, carboxyl acyl aminotriazole, CM101, Marimastat, pentosane polysulfate ester, angiopoietin-2, interferon-' alpha ', Antibiotic TAN 420F, PNU145156E, 16K lactotropin fragment, linomide (Roquinimex), Thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 and minocycline.

12. the method for claim 1, the aminoacid sequence choosing of wherein said AD part is the following group forming freely: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID N0:81, SEQ ID NO:82, SEQ ID NO:83 and SEQ ID NO:84.

13. the method for claim 1, the aminoacid sequence choosing of wherein said DDD part is the following group forming freely: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:31.

14. the method for claim 1, wherein said interferon-antibody complex is more effective than the combination of independent described interferon, independent described antibody or interferon that yoke does not close and antibody.

15. the method for claim 1, wherein said cancer choosing is the following group forming freely: acute lymphoblastic leukemia, acute myeloid leukemia, gallbladder cancer, breast carcinoma, cervical cancer, chronic lymphocytic leukemia, chronic granulocytic leukemia, colorectal carcinoma, carcinoma of endometrium, esophageal carcinoma, gastric cancer, head and neck cancer, Hodgkin lymphoma, pulmonary carcinoma, medullary thyroid carcinoma, non-Hodgkin lymphoma, multiple myeloma, renal carcinoma, ovarian cancer, cancer of pancreas, glioma, melanoma, hepatocarcinoma, carcinoma of prostate and bladder cancer.

16. the method for claim 1, wherein said viral infection choosing is the following group forming freely: HIV (human immunodeficiency virus) (HIV), herpesvirus, herpes simplex virus, vaccinia virus, cytomegalovirus, rabies virus, influenza virus, rhinovirus, hepatitis B virus, hepatitis C virus, Sendai virus, feline leukaemia virus, reovirus, poliovirus, the tiny sample virus of serum human, simian virus 40, respiratory syncytial virus, mouse mammary tumor virus, varicella zoster virus, dengue virus, rubella virus, Measles virus, adenovirus, human T-leukemia virus, epstein-Barr virus, muroid leucovirus, mumps virus, vesicular stomatitis virus, sindbis alphavirus, lymphocytic choriomeningitis virus, Verrucosis poison and blue tongue rims.

17. methods as claimed in claim 16, wherein said viral infection is chronic hepatitis C infection.

18. the method for claim 1, wherein said antibody is anti-HIV antibody, anti-gp120 antibody, anti-gp41 antibody, HCV antigen/antibody combination, malaria antibody, protozoacide antibody, schistosomicide antibody, anti-trypanosomicide antibody, anti bacterial antibody, antiviral antibody and anti fungal antibody.

19. the method for claim 1, wherein said disease is asthma or multiple sclerosis.

20. methods as claimed in claim 19, wherein said disease is that asthma and described complex are used by nose.

21. methods as claimed in claim 20, wherein said antibody or antibody fragment are anti-TNF, anti-CD74 or anti-MIF antibody or antibody fragment.

22. 1 kinds of methods for the treatment of cancer, viral infection, asthma or multiple sclerosis, it comprises:

A) obtain interferon-antibody complex, it comprises:

I) the first fusion rotein, it comprises dimerization and stop domain (DDD) interferon-λ partly that is connected to human kinase protein A (PKA) RI, RI β, RII or RII β;

Ii) the second fusion rotein, the antibody that it comprises the anchoring structure territory (AD) that is connected to AKAP protein or antigen binding antibody fragment; And

B) to the experimenter with cancer, viral infection, asthma or multiple sclerosis, use described interferon-antibody complex;

Wherein the described DDD of two copies partly forms dimer, and described dimer is incorporated into described AD part to form described complex.

23. 1 kinds of methods for the treatment of cancer, viral infection, asthma or multiple sclerosis, it comprises:

A) to the experimenter with cancer, viral infection, asthma or multiple sclerosis, use bi-specific antibody complex, described complex comprises: (i) the first fusion rotein, and it comprises dimerization and stop domain (DDD) first antibody or its Fab partly that is connected to human kinase protein A (PKA) RI, RI β, RII or RII β; (ii) the second fusion rotein, the second antibody that it comprises the anchoring structure territory (AD) that is connected to AKAP protein or its antigen binding antibody fragment, wherein said the first and second antibody or its fragment are incorporated into disease association antigen and are incorporated into hapten; And

B) to described experimenter use comprise the described haptenic of interferon-λ and at least one copy can targeting construct, wherein saidly can be incorporated into described bi-specific antibody to send described interferon-λ to diseased cells by targeting construct.

24. 1 kinds of compositionss, it comprises:

A) the first fusion rotein, interferon-λ that it comprises anchoring structure territory (AD) part that is connected to AKAP protein; With

B) the second fusion rotein, it comprises the dimerization that is connected to human kinase protein A (PKA) RI, RI β, RII or RII β and antibody or the antigen binding antibody fragment of stopping domain (DDD) part; And

Wherein the described DDD of two copies partly forms dimer, and described dimer is incorporated into described AD part to form described complex.

25. compositionss as claimed in claim 24, the group that wherein said interferon-λ selects free human interferon-λ 1, interferon-λ 2 and interferon-λ 3 to form.

26. compositionss as claimed in claim 24, wherein said antibody or antibody fragment are incorporated into the freely antigen of the following group forming of choosing: carbonic anhydrase IX, CCCL19, CCCL21, CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, AFP, PSMA, CEACAM5, CEACAM-6, B7, the ED-B of fibronectin, factor H, FHL-1, Flt-3, folacin receptor, GROB, HMGB-1, hypoxia inducible factor (HIF), HM1.24, insulin-like growth factor-i (ILGF-1), IFN-, IFN-, IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, PAM4 antigen, NCA-95, NCA-90, Ia, HM1.24, EGP-1, EGP-2, HLA-DR, tenascin, Le (y), RANTES, T101, TAC, Tn antigen, Thomson-Friedenreich antigen, neoplasm necrosis antigen, TNF-, TRAIL receptor (R1 and R2), VEGFR, EGFR, PlGF, complement factor C3, C3a, C3b, C5a, C5 and oncogene products.

27. compositionss as claimed in claim 24, wherein said antibody or its antibody fragment are incorporated into the freely antigen of the following group forming of choosing: TROP-2, CEACAM5, CEACAM6, HLA-DR, CD19, CD20, CD22, CD74, CD52 and EGFR.

28. compositionss as claimed in claim 24, the group that wherein said antibody or its antibody fragment select free hLL1, hLL2, RFB4, hRS7, hPAM4, hMN-3, hMN-14, hMN-15, hMu-9, Immu-31, hL243, hA19, hA20 and hR1 to form.

29. compositionss as claimed in claim 24, the group that wherein said antibody fragment selects free Fab, Fv, scFv and dAb to form.

30. compositions as claimed in claim 24, it further comprises the freely other treatment agent of the following group forming of choosing: second antibody, second antibody fragment, medicine, toxin, enzyme, hormone, immunomodulator, cytokine, chemotactic factor, antisense oligonucleotide, siRNA, RNAi, radionuclide, boron compound, photosensitizer, anti-angiogenic agent and short apoptosis agent.

31. compositionss as claimed in claim 30, wherein said medicine choosing is the following group forming freely: 5-fluorouracil, APL, azaribine, Anastrozole, anthracycline, bendamustine, bleomycin, bortezomib, Bruton inhibitors of kinases, Bryostatin-1, busulfan, calicheamycin, camptothecine, carboplatin, 10-hydroxycamptothecine, carmustine, celecoxib, chlorambucil, cisplatin (CDDP), Cox-2 inhibitor, irinotecan (CPT-11), SN-38, carboplatin, carat Qu Bin, camptothecin, cyclophosphamide, cytosine arabinoside, dacarbazine, Docetaxel, dactinomycin, daunorubicin, amycin, 2-pyrrolinyl amycin (2P-DOX), cyano group-morpholinyl amycin, amycin glucosiduronic acid, epirubicin glucosiduronic acid, estramustine, table podophyllotoxin, estrogen receptor bonding agent, etoposide (VP16), etoposide glucosiduronic acid, etoposide phosphate, floxuridine (FUdR), 3 ', 5 '-O-dioleoyl-FudR, fludarabine, flutamide, farnesyl-protein transferase inhibitor, gemcitabine, hydroxyurea, idarubicin, ifosfamide, leunase, lenalidomide, formyl tetrahydrofolic acid, lomustine, chlormethine, melphalan, purinethol, Ismipur, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, nvelbine, nitroso ureas, plicamycin, procarbazine, paclitaxel, pentostatin, PSI-341, raloxifene, semustine, streptozotocin, tamoxifen, taxol, temozolomide, anti-platinum, Thalidomide, thioguanine, thio-tepa, teniposide, topotecan, tyrosine kinase inhibitor, uracil mustard, vinorelbine, vinblastine, vincristine, vinca alkaloids, LFM-A13, Dasatinib, imatinib or AMN107.

32. compositionss as claimed in claim 30, the group that wherein said toxin selects free Ricin, abrin, alpha toxin, saporin, ribonuclease (RNA enzyme), ranpirnase, DNA enzyme I, staphylococcus enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, diphtherotoxin, Rhodopseudomonas extracellular toxin and Rhodopseudomonas endotoxin to form.

33. compositionss as claimed in claim 30, wherein said anti-angiogenic agent choosing is the following group forming freely: angiostatin, bar chalone, canstatin, mammary gland silk presses down albumen, VEGF antibody, anti-PlGF antibody, laminin，LN peptide, fibronectin, plasminogen activator inhibitor, tissue inhibitor of metalloproteinase, interferon, interleukin 12, IP-10, Gro-β, thrombospondin, 2ME2, proliferin associated protein, carboxyl acyl aminotriazole, CM101, Marimastat, pentosane polysulfate ester, angiopoietin-2, interferon-' alpha ', Antibiotic TAN 420F, PNU145156E, 16K lactotropin fragment, linomide (Roquinimex), Thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 and minocycline.

34. compositionss as claimed in claim 24, the aminoacid sequence choosing of wherein said AD part is the following group forming freely: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID N0:81, SEQ ID NO:82, SEQ ID NO:83 and SEQ ID NO:84.

35. compositionss as claimed in claim 24, the aminoacid sequence choosing of wherein said DDD part is the following group forming freely: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:31.

36. compositionss as claimed in claim 24, wherein said interferon-antibody complex is more effective than the combination of independent described interferon, independent described antibody or interferon that yoke does not close and antibody.

37. 1 kinds of compositionss, it comprises:

A) the first fusion rotein, it comprises dimerization and stop domain (DDD) interferon-λ partly that is connected to human kinase protein A (PKA) RI, RI β, RII or RII β; With

B) the second fusion rotein, antibody or antigen binding antibody fragment that it comprises anchoring structure territory (AD) part that is connected to AKAP protein;

Wherein the described DDD of two copies partly forms dimer, and described dimer is incorporated into described AD part to form described complex.