CN104159600B - Effectively strengthen antitumor and antiviral activity with antibody target interferon lambda - Google Patents

Abstract

The present invention relates to the method and composition for forming interferon lambda and antibody or the compound of antigen binding antibody fragment.In preferred embodiments, the interferon lambda and the antibody or fragment are fusion proteins, each self-contained dimerization and dockerin domain (DDD) part from human kinase protein A or anchoring domain (AD) part from A kinase anchoring proteins (AKAP).In a more preferred embodiment, the combination of the interferon antibody compound than single interferon lambda, single antibody or not conjugated interferon lambda and antibody is more efficiently used for treating cancer, asthma, Alzheimer's (Alzheimer ' s disease), multiple sclerosis or virus infection.

Description

Effectively strengthen antitumor and antiviral activity with antibody target interferon-λ

Related application

The application require that the Provisional U.S. Patent Application 61/ submitted on January 26th, 2012 according to 35U.S.C.119 (e) The rights and interests for the U.S. Patent application No.13/412,816 that on March 6th, 591,087 and 2012 submits.Above-cited each preferential Shen Text please is incorporated herein in entirety by reference.

Sequence table

The application includes the sequence table submitted by EFS-Web and be incorporated in entirety by reference hereby.The ASCII For copy creating on January 22nd, 2013, it was 50,753 bytes to be named as IBC136WO.txt and size.

Background

Invention field

The present invention relates to include the interferon-λ (IFN- λ), more preferably for being connected to antibody or antigen binding antibody fragment IFN- λ 1 DOCK-AND-LOCK^TM(DNL^TM) compound composition and treatment application 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.However, it would be recognized by those skilled in the art that the present invention is not so limited and more broadly contained Lid antibody-interferon compound.Preferably, the DNL^TMCompound is to use such as United States Patent (USP) No.7,521,056,7,527, 787th, 7,534,866,7,550,143 and 7, made from the composition and technology shown in 666,400, the patent is respective Embodiment part is incorporated herein by reference.The conjugated interferon of antibody, which retains external activity and shown, generally to be strengthened Inside effect and increased serum half-life.DNL^TMOther advantages of product may also include relatively low immunogenicity, administration frequency Rate reduces, dissolubility increases, stability enhancing and renal clearance reduce.

Background context

It is reported that interferon-' alpha ' (IFN α) is in animal cancer models (Ferrantini etc., 1994, J Immunol153： 4604-15) and human patients with cancer (Gutterman etc., 1980, Ann Intern Med93：All have in 399-406) anti-swollen Tumor activity.IFN α can play a variety of direct antitumor actions, including lower oncogene, up-regulation tumor suppressor, pass through Increase the expression of tumor surface MHC I albuminoids and strengthen Immune discrimination, strengthen Apoptosis, and the increasing to chemotherapeutant Quick (Gutterman etc., 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 tumours, IFN α can by activate STAT1 and with directly 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 be most important to overall antitumor reaction but largely underestimated (Belardelli etc., 1996, Immunol Today17：369-72).IFN α by bone marrow 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, Immunity14：Effect 461-70) and there is pleiotropism influence on immune response.Important tune as innate immune system Save agent, the quick differentiation of IFN α inducing dendritic cell and activation (Belardelli etc., 2004, Cancer Res64：6827-30； Paquette etc., 1998, J Leukoc Biol64：358-67；Santini etc., 2000, J Exp med191：1777-88) simultaneously Strengthen cytotoxicity, migration, cell factor generation and NK cells antibody-dependent cytotoxicity (ADCC) (Biron etc., 1999, Annu Rev Immunol17：189-220；Brunda etc., 1984, Cancer Res44：597-601).

So far, it is used to treating hairy cell leukemia, chronic granulocytic leukemia, pernicious black by ratifying IFN-α 2 Melanoma, follicular lymphoma, condyloma acuminatum, AID correlations Kaposi sarcoma (AIDs-related Kaposi Sarcoma), chronic hepatitis B and hepatitis C；IFN-β is used to treat multiple sclerosis；And IFN-γ is used to treat Chronic granulo matosis and pernicious osteopetrosis, it was verified that IFN treatment validity.Although on this group of autocrine and other point Secrete that the document of cell factor is numerous, but its function in health and disease is still elucidated with, including what is clinically introduced more have The form of effect and novelty (Pestka, 2007, J.Biol.Chem282：20047-51；Vilcek, 2006, Immunity25： 343-48)。

Interferon plays a key effect in antitumor and antimicrobial host defense, and is explored conduct extensively The therapeutic agent of cancer and infectious diseases (Billiau etc., 2006, Cytokine Growth Factor Rev17：381-409； Pestka etc., 2004, Immunol Rev202：8-32).Although I types and II types interferon (IFN-α/β and γ) have been made quite Big effort, but due to its short circulating half-life in patients, general toxicity and not good enough reaction and in clinic configures Use be limited (Pestka etc., 2004, Immunol Rev202：8-32；Miller etc., 2009, Ann N Y Acad Sci1182：69-79).IFN- λ families is found to be exploitation for the optional of these unsatisfied clinical indications at the beginning of 2003 IFN agent brings-individual exciting new chance (Kotenko etc., 2003, Nat Immunol4：69-77；Sheppard etc., 2003, Nat Immunol4：63-8).

IFN- λ (be referred to as type iii interferon) be one group of newest description by IFN- λ 1,2,3 (also referred to as interferon- 29th, 28A and 28B) composition cell factor, they by chromosome 19 three different genes carry out genetic coding (Kotenko etc., 2003, Nat Immunol4：69-77；Sheppard etc., 2003, Nat Immunol4：63-8).In albumen In matter level, IFN- λ 2 and the very high homologies of IFN- λ 3 have 96% amino acid identities, and IFN- λ 1 and IFN- λ 2 and IFN- λ 3 Shared about 81% homology (Sheppard etc., 2003, Nat Immunol4：63-8).IFN- λ pass through with being induced by I types IFN Similar JAK/STAT pathway activation signal transductions, including the activation of JAK1 and TYK2 kinases, the phosphorylation of stat protein and Activation (Witte etc., 2010, Cytokine Growth of the transcription complex for the gene factor 3 (ISGF3) that IFN is stimulated Factor Rev21：237-51；Zhou etc., 2007, J Virol81：7749-58).

Main Differences between type III IFN systems and I type IFN systems are the distribution of its respective receptor complex. IFN-α/β carries out signal transduction by the I types interferon receptors of two kinds of wide expressions, and applies relevant institute with IFN-α/β Obtain general toxicity and limit their use (Pestka etc., 2007, J Biol Chem282 as therapeutic agent：20047- 51).By contrast, IFN- λ the IFN- λ acceptors 1 (IFN- λ R1) and IL-10 acceptors 2 (IL-10R2) of uniqueness by being made up of Heterodimeric receptor complex carries out signal transduction.(Witte etc., 2009, Genes Immun10 as reported previously：702- 14), IFN- λ R1 have an expression pattern being very limited, wherein in epithelial cell, melanocyte and liver cell it is horizontal most Height, and it is horizontal minimum in primary central nervous system (CNS) cell.The expression of haematogenic immunity system cells is high-caliber short IFN- λ acceptors splice variant (sIFN- λ R1), it suppresses IFN- λ effects.The finite response of neuronal cell and immunocyte It is meant that using IFN- λ can make to IFN-α therapy frequently related serious toxicity be not present or substantially reduce (Witte etc., 2009, Genes Immun10：702-14；Witte etc., 2010, Cytokine Growth Factor Rev21：237-51). New publication is reported, although IFN-α and IFN- λ induce table of one group of common ISG (interferon-stimulated gene) in liver cell Reach, but it is different from IFN-α, and IFN- λ administration will not induce STAT activation or ISG in the lymphocyte or monocyte of purifying (12/20/12) Dickensheets etc., 2013, J Leukoc Biol.93, is published on the net for expression.This shows for chronic The treatment of HCV infection, IFN- λ may be better than IFN-α, because to be less likely induction often related to IFN-α therapy white by IFN- λ Leukopenia (Dickensheets etc., 2013).

IFN- λ show architectural feature as the cytokine class relevant with IL-10, but functionally there is I type IFN classes to resist Virus and antiproliferative activity (Witte etc., 2009, Genes Immun10：702-14；Ank etc., 2006, J Virol80：4501- 9；Robek etc., 2005, J Virol79：3851-4).IFN- λ 1 and IFN- λ 2 are verified to reduce virus replication or various diseases The cytopathic effect of poison, the virus include DNA virus (hepatitis type 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 virus (EMCV；Sheppard etc., 2003, Nat Immunol4：63-8) and HCV (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 virus (vesicular stomatitis virus；Pagliaccetti etc., 2008, J Biol Chem283：30079-89) and Flu-A Virus (Jewell etc., 2010, J Virol84：11515-22) and diplornavirus such as rotavirus (Pott etc., 2011, PNAS USA108：7944049).IFN- λ 3 have been accredited as the key cytokines (Ge in HCV infection according to genetic research Deng 2009, Nature461：399-401), and also shown for EMCV lateral reactivity (Dellgren etc., 2009, Genes Immun10：125-31).Lack caused by the IFN- λ of rhinovirus induction it is reported that aggravating with the asthma that rhinovirus induces Order of severity height correlation (Contoli etc., 2006, Nature Med12：1023-26) and IFN- lambda therapies have been proposed New method (Edwards and Johnston, 2011, EMBO Mol Med3 as treatment allergic asthma：306-8； Koltsida etc., 2011, EMBO Mol Med3：348-61).

IFN- λ antiproliferative activity, including neuroendocrine carcinoma have been determined in several human cancer cell lines BON1 (Zitzmann etc., 2006,344：1334-41), glioblastoma LN319 (Meager etc., 2005, Cytokine31： 109-18), immortalized keratinocytes HaCaT (Maher etc., 2008, Cancer Biol Ther7：1109-15), black Plain knurl F01 (Guenterberg etc., 2010, Mol Cancer Ther9：510-20) and cancer of the esophagus TE-11 (Li etc., 2010, Eur J Cancer46：180-90).In animal model, IFN- λ are thin by the congenital and reaction induced tumour of adaptive immunity Born of the same parents' apoptosis and destruction, this shows that IFN- λ local delivery is probably the useful auxiliary strategy in human malignancies treatment (Numasaki etc., 2007, J Immunol178：5086-98).

In clinic configures, Pegylation IFN- λ 1 (PEG-IFN- λ 1) have been temporarily used for chronic hepatitis liver The patient of scorching virus infection.In the Ib stages study (n=56), all observed under all dosage levels (0.5-3.0 μ g/kg) To antiviral activity, and when PEG-IFN- λ 1 to be applied to the 1 type HCV patient of gene recurred after IFN-α therapy, virus Carrying capacity from 2.3 be down to 4.0log (Muir etc., 2010, Hepatology52：822-32).The IIb stages study (n=526) display Patient with the type of gene 1 and 4 type HCV is treated with significantly higher for being treated with PEG-IFN- λ 1 compared to PEG-IFN- α Reactivity.At the same time, using PEG-IFN- λ 1 situation compared to using in the case of PEG-IFN- α with I type interferon The generally related adverse events incidence for the treatment of is relatively low.Neutrophilic granulocytopenia and decrease of platelet are seldom observed, and is flowed The incidence of sense sample symptom, anaemia and musculoskeletal symptom is down to about 1/3 seen by PEG-IFN- α treatments.However, PEG- Serious adverse events, depression and other common adverse events (>=10%) incidences between IFN- λ 1 and PEG-IFN- α It is similar.Compared with PEG-IFN- α, the hepatotoxicity wind agitation of visible more height ratio in maximum dose level PEG-IFN- λ 1 (“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, " on April 2nd, 2011, Press Release from Bristol-Myers Squibb).

The composition and method for including interferon-λ-antibody complex are needed, the compound keeps unmodified interferon Bioactivity, but show improved in vivo efficacy, reduce toxicity and/or excellent pharmacokinetic property.

The content of the invention

The invention discloses for producing DNL^TMThe method and composition of compound, the compound include and are connected to antibody Or the interferon of antigen binding antibody fragment, preferably interferon-λ, more preferably IFN- λ 1.The interferon part can be conjugated to people Albuminoid kinases A (PKA) regulation subunits RI α, RI β, RII α or RII β dimerization and dockerin domain (DDD) part or optional Conjugated anchoring domain (AD) part to A kinase anchoring proteins (AKAP) in ground.Antibody or antibody fragment portion are conjugated to complementary Property AD or DDD parts.Because DDD parts spontaneously form dimer, the dimer is incorporated into AD parts, institute with high-affinity So that DNL can be filled by the effect such as interferon, antibody or antibody fragment subgroup^TMCompound, the effector are each attached to DDD Part or AD parts., can be by substantially by the way that the effector for being connected to DDD parts is mixed with being connected to the effector of AD parts Any effector is incorporated to DNL^TMIn compound.

DNL^TMCompound preferably comprises one, the interferon part for being connected to antibody or fragment of two or four copy. However, it would be recognized by those skilled in the art that can be built in the range of method and composition claimed and using tool There is the other kinds of DNL of different structures and different interferon/antibody ratio^TMCompound, such as United States Patent (USP) No.7,521, 056th, disclosed in 7,527,787,7,534,866,7,550,143 and 7,666,400.In a more preferred embodiment, may be used The disulfide bond of compound can be stabilized to be formed by introducing cysteine residues by appropriate location in DDD and AD sequences, from And by DNL^TMCompound covalence stablility.

In other preferred embodiments, the DNL of the conjugated antibody of interferon is included^TMCompound is shown than not conjugated interference The serum clearance rate of plain slow at least one order of magnitude.

It would be recognized by those skilled in the art that the technology is not limited to interferon-λ, but it can be applied to a variety of therapeutic agents Targeted delivery, the therapeutic agent include but is not limited to enzyme, cell factor, chemotactic factor (CF), growth factor, peptide, fit, blood red egg In vain, antibody and its fragment.Exemplary dose include MIF, HMGB-1 (High mobility group box-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- 78th, MCP-1, IP-10, Gro- β, ECF (Eotaxin), interferon-' alpha ', interferon-beta, interferon- γ, interferon-λ, G-CSF, GM-CSF, SCF, PDGF, MSF, Flt-3 part, hematopoietin (erythropoietin), TPO (thrombopoietin), hGH, CNTF, leptin (leptin), oncostatin M, VEGF, EGF, FGF, PIGF, insulin, hGH, calcitonin, Factor IX, IGF, growth hormone release inhibiting hormone, tissue plasminogen activator And LIF.

Antibody available for the targeting cancer therapy in the range of method and composition claimed includes (but unlimited In) LL1 (anti-CD74), LL2 and RFB4 (anti-CD22), RS7 (anti-Glycoproteins in Epithelial -1 (EGP-1)), PAM4 and KC4 (be anti- Mucoprotein), MN-14 (anti-carcinoembryonic antigen (CEA, also referred to as CD66e), MN-15 (anti-CEACAM6), Mu-9 (anti-colon-specifics Antigen-p), Immu-31 (Anti-α-Fetoprotein), anti-TAG-72 (such as CC49), (anti-PSMA is (preceding by anti-Tn, J591 or HuJ591 Row gland specific membrane antigen)), AB-PG1-XG1-026 (anti-PSMA dimers), D2/B (anti-PSMA), G250 (anti-carbonic anhydrases IX), hL243 (anti-HLA-DR), alemtuzumab (alemtuzumab) (anti-CD52), bevacizumab (bevacizumab) are (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 toltrazuril Monoclonal antibody (trastuzumab) (anti-ErbB).These antibody be it is as known in the art (for example, United States Patent (USP) No.5,686, 072、5,874,540、6,107,090、6,183,744、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；And U.S. Patent Application Publication No.20040202666 (abstention), 20050271671 and 20060193865；The embodiment part of each document is to quote Mode is incorporated herein).Specific known antibodies used include hPAM4 (United States Patent (USP) No.7,282,567), hA20, and (U.S. is special Sharp No.7,251,164), hA19 (United States Patent (USP) No.7,109,304), hIMMU31 (United States Patent (USP) No.7,300,655), hLL1 (United States Patent (USP) No.7,312,318), hLL2 (United States Patent (USP) No.7,074,403), hMu-9 (United States Patent (USP) No.7,387, 773), hL243 (United States Patent (USP) No.7,612,180), hMN-14 (United States Patent (USP) No.6,676,924), hMN-15 (United States Patent (USP)s No.7,541,440), hR1 (U.S. Patent application 12/772,645), hRS7 (United States Patent (USP) No.7,238,785), hMN-3 are (beautiful State patent No.7,541,440), AB-PG1-XG1-026 (U.S. Patent application 11/983,372, with ATCC PTA-4405 and PTA-4406 is deposited) and D2/B (WO2009/130575), each the patent or the text of application are with drawings and examples The mode that part is quoted is incorporated herein.

Anti-TNF-α antibody is as known in the art and available for treating immunological diseases, such as asthma (see, for example, Erin etc., 2006, Am J Respir Crit Care Med174：753-62).The known antibody for TNF-α includes people Antibody-like 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 it is many other Know that anti-TNF-α antibody can be used in method and composition claimed.Other antibody used include but is not limited to anti-B Cell antibody, such as dimension trastuzumab (veltuzumab), epratuzumab (epratuzumab), meter La Zhu monoclonal antibodies Or hL243 (milatuzumab)；Torr pearl monoclonal antibody (tocilizumab) (anti-IL-6 acceptors)；Basiliximab (basiliximab) (anti-CD25)；Daclizumab (daclizumab) (anti-CD25)；Efalizumab (efalizumab) (anti-CD11a)；Muromonab (muromonab)-CD3 (AntiCD3 McAb acceptor)；Anti-CD 40 L (UCB, Brussels, Belgium)；That His pearl monoclonal antibody (natalizumab) (integrin of anti alpha 4) and omalizumab (omalizumab) (anti-IgE).

Macrophage migration inhibitory factor (MIF) be congenital and adaptive immunity and Apoptosis important regulation because Son.It has been reported that CD74 is MIF endogenous recipient (Leng etc., 2003, J Exp Med197：1467-76).Antagonism resists Therapeutic action of the CD74 antibody for the MIF intracellular pathways mediated can be used for treating extensive various disease states, such as bladder Cancer, prostate cancer, breast cancer, lung cancer, colon cancer and chronic lymphocytic leukemia (for example, Meyer-Siegler etc., 2004, BMC Cancer12：34；Shachar and Haran, 2011, Leuk Lymphoma52：1446-54)；Nephrosis, such as kidney Dirty allograft rejection reacts (Lan, 2008, Nephron Exp Nephrol.109：e79-83)；And numerous hair (Meyer-Siegler etc., 2009, Mediators Inflamm, electronic publishing was on March 22nd, 2009 for inflammatory disease； Takahashi etc., 2009, Respir Res10：33).Meter La Zhu monoclonal antibodies (hLL1) are that have to be used to treat the mediated diseases of MIF Therapeutical uses exemplary anti-CD74 antibody.

The pharmaceutical composition of the present invention can be used for treatment tested with neurodegenerative disorders such as Alzheimer's Person.A bar pearl monoclonal antibody (bapineuzumab) is in the clinical test for the treatment of of alzheimer's disease.It is proposed to be used in A Er Other antibody of thatch sea Mo's disease treatment include Alz50 (Ksiezak-Reding etc., 1987, J Biol Chem263：7943- 47), more spit of fland reed monoclonal antibody (gantenerumab) and Su Lan pearls monoclonal antibody (solanezumab).A kind of infliximab (anti-tnf-alpha Antibody) being reported to reduce amyloid patch and improve recognizes.The neuron of Alzheimer patients' brain CD74 presence (Bryan etc., Mol Neurodegeneration2008 in fibre matting；3：13doi：10.1186/1750- It can be the target of peptide or antibody therapy 1326-3-13) to show CD74, or for making therapeutic agent target Alzheimer patients These regions of brain.

Other workable antibody include being directed to infectious diseases pathogen such as bacterium, virus, mycoplasma or other cause of diseases The antibody of body.Many is as known in the art and any this known antibody for the antibody of these infectious pathogens It can be used in method and composition claimed.For example, the gp120 sugar for human immunodeficiency virus I (HIV-1) The antibody of proteantigen is known, and some this antibody can have immanoprotection action in the mankind.See, for example, Rossi etc., Proc.Natl.Acad.Sci.USA.86：8055-8058,1990.Known ANTI-HIV DRUGS include by (AIDS.2006 October 3 such as Johansson；20(15)：1911-5) the anti-anti-envelope antibodies of description, and by Polymun (Vienna, Austria) is described and the ANTI-HIV DRUGS of sale, and 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, all it is incorporated herein by reference.

Zygoblast, merozoite, schizont and gametophyte stage can be directed to for the antibody of malarial parasite.Pin is generated To the monoclonal antibody of zygoblast (Circumsporozoite antigen), and shown its in vitro with zygoblast can be neutralized in rodent (N.Yoshida etc., Science207：71-73,1980).Some team have developed toxoplasma antibody, and Infection of Toxoplasma Gondii is arch Involved protozoon parasite (Kasper etc., J.Immunol.129 in parasitosis：1694-1699,1982；Ibid, 30： 2407-2412,1983).Developed the antibody for virgin worm (schistosomulae) surface antigen and have found its It is inner or in vitro act on 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 American trypanosomiasis (Chagas ' disease) pathogen, and Stinkbug entomochory is hunted by what is sucked blood.Generate specificity in vitro and suppress a kind of form of parasite to another form The differentiation of (epimastigote to trypomastigote stage) and the antibody reacted with cell surface glycoprotein；However, the food in one's mouth of parasite Newborn animal (blood flow) form be not present this antigen (Sher etc., Nature, 300：639-640,1982).

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

It is (bacterium, virus, primary dynamic the microorganism for causing largely to infect in the mankind for most of has been developed Thing, fungi, other parasites) suitable antibodies, and many be previously used for in-vitro diagnosis purpose.Commercially available is directed to The antibody of extensive multiple pathogens is known and can be utilized.From the exemplary of Millipore (Billerica, MA) Anti-pathogen antibodies include anti-Escherichia coli (E.coli) antibody (MAB8272), anti-salmonella category (Salmonella) antibody (MAB748-MG-K), anti-listeria monocytogenes (Listeria monocytogenes) antibody (MAB8001), Helicobacter pylori resistant (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-mouse giardia lamblia stiles (Giardia Muris) antibody (MAB10242), anti-dengue virus (Dengue virus) antibody (MAB10226), anti-flavivirus category (Flavivirus) antibody (MAB10216), anti-legionella pneumophilia (L.pneumophila) antibody (MAB10223), anti-B-mode liver Scorching antibody (MAB10201), anti-dengue virus antibody (MAB10217), anti-herpes simplex antibody (MAB8685), anti-Ai Posi Smooth epstein-Barr virus (Epstein Barr virus) antibody (MAB10219), anti-Coxsackie virus (Coxsackie virus) are anti- Body (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-nipple Shape tumor virus (Papillomavirus) antibody (MAB837), anti-HCV (AB307), anti-enteric virus71 antibody (MAB979-K), anti-measles antibody (MAB8905-K), anti-cytomegalovirus (Cytomegalo virus) antibody (MAB8140- KC), viral (the Yellow Fever virus) antibody (MAB984-K) of yellow fever, anti-rabies antibody (MAB8724), anti-blister Viral (Poliovirus) antibody (MAB8566) of the antibody of exanthema virus 6 (MAB8535), poliomyelitis, 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's (Santa Cruz, CA) Exemplary anti-pathogen antibodies include anti-dengue virus antibody (sc-325018), anti-vaccinia virus antibody (sc-69949), resisted Polyomavirus 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- Hantaan virus (Hantavirus) antibody (sc-57755) and anti-mycoplasma hominis (Mycoplasma hominis) virus (sc-58171).Exemplary anti-pathogen antibodies from ProSci Inc. (Poway, CA) include anti-Eurotium (Aspergillus) antibody (35-595), anti-candida albicanses (Candida albicans) antibody (35-121) and anti-wine brewing Yeast (Saccharomyces cerevisiae) antibody (35-361).It is exemplary anti-from KPL (Gaithersburg, MD) Pathogen antigen includes anti-Staphylococcus aureus antibody (01-90-05), anti-lyme disease spirochete (Borrelia Burgdorferi) antibody (01-97-91), helicobacter pylori resistant antibody (01-93-94), anti-Legionella category (Legionella Spp.) antibody (01-90-03) and anti-yersinia's genus (Yersinia spp.) antibody (01-90-04).Many this areas be present In other well-known sources business anti-pathogen antibodies.It would be recognized by those skilled in the art that for substantial any institute The antibody of pathogen interested is commercially available and/or disclosure obtains, and any such known antibody may be incorporated into use The DNL of methods described below and composition^TMIn compound.

The DNL^TMCompound is suitable for a variety of treatments and diagnostic application extensively.DNL^TMThe application method of compound can Including detecting, diagnosing and/or treat disease or other Medical Conditions.These symptom may include (but not limited to) cancer, hyperplasia, heavy breathing Asthma, multiple sclerosis, infectious diseases, chronic viral hepatitis, herpesvirus infection, chronic type b or HCV Infection, chronic granulo matosis, pernicious osteopetrosis, Kaposi sarcoma (Karposi ' s sarcoma), human papilloma disease Malicious infection, influenza, chronic granulocytic leukemia, hairy cell leukemia, skin T cell lymphoma, follicular lymphoma, transfer Property clear-cell carcinoma, hemangioma, hematology's malignant tumour, condyloma acuminatum and malignant mela noma.

Medicable exemplary oncologic type include acute lymphoblastic leukemia, acute myelocytic leukemia, Courage cancer, breast cancer, cervix cancer, chronic lymphocytic leukemia, chronic granulocytic leukemia, colorectal cancer, uterus Endometrial carcinomas, cancer of the esophagus, stomach cancer, head and neck cancer, Hodgkin lymphoma (Hodgkin ' s lymphoma), lung cancer, medullary carcinoma of thyroid gland, NHL, Huppert's disease, kidney, oophoroma, cancer of pancreas, glioma, melanoma, liver cancer, forefront Gland cancer and carcinoma of urinary bladder.

Various virus infection can be treated, including but not limited to human immunodeficiency virus (HIV), herpesviral, simple Herpesviral, vaccinia virus, cytomegalovirus, rabies viruses, influenza virus, rhinovirus, hepatitis type B virus, hepatitis C virus Poison, sendai virus (Sendai virus), feline leukaemia virus, reovirus (Reo virus), poliovirus, The tiny sample virus of human serum, simian virus 40, Respiratory Syncytial Virus(RSV), mouse mammary tumor virus, varicella-zoster disease Poison, dengue virus, rubella virus, measles virus, adenovirus, human T-leukemia virus, epstein-Barr virus, mouse Quasi-leukemia virus, mumps virus, vesicular stomatitis virus, sindbis alphavirus (Sindbis virus), lymphatic Choriomeningitis virus, verrucosis poison and blue tongue rims；Or bacterium is selected from the group consisted of：Streptococcusagalactiae (Streptococcus agalactiae), legionella pneumophilia (Legionella pneumophila), micrococcus scarlatinae (Streptococcus pyogenes), Escherichia coli (Escherichia coli), NEISSERIA GONORRHOEAE (Neisseria Gonorrhoeae), Neisseria meningitidis (Neisseria meningitidis), pneumococcus (Pneumococcus), B-mode Haemophilus influenzae (Hemophilus influenzae B), treponemal bacterium (Treponema pallidum), Lyme disease spiral shell Revolve body (Lyme disease spirochetes), pseudomonas aeruginosa (Pseudomonas aeruginosa), leprosy branch Bacillus (Mycobacterium leprae), Bacillus abortus (Brucella abortus), mycobacterium tuberculosis (Mycobacterium tuberculosis) or clostridium tetani (Clostridium tetani).

Brief Description Of Drawings

Fig. 1 is the gene structure (A and B) for the expression for describing cell factor-DDD2 (C) and IgG-AD2 (D) DNL modules Cartoon figure.The module is combined to form the DNL structures being made up of four cell factors for being fused to IgG (E).

Fig. 2 shows that external IFN α of the cell factor-MAb DNL constructs compared with Pegylation or primary IFN α is lived Property.Measurement activity specific (IU/pmol) as be shown in the examples.From each of rhIFN α 2b standard curves extrapolation concentration known The activity of test article.20-2b (●), 734-2b (■), v-mab (zero), v-mab+734-2b () in progressive concentration, PEGASYS, PEG- intrones (▲) or 1R-2bIn the presence of culture is grown and relatively living thin with MTS measurements Born of the same parents' density.The % of the signal obtained from untreated cell is drawn relative to the curve of the log values of molar concentration.Use Prism Software Create dose-response curve and EC₅₀Value.Error bars, SD.

Reporter determination methods of the Fig. 2 (A) based on cell.

Fig. 2 (B) utilizes EMC viruses and the virus protection determination method of A549 cells.

Fig. 2 (C) uses the external lymthoma proliferation assay of Daudi cells.

Fig. 2 (D) uses the external lymthoma proliferation assay of Jeko-1 cells.

Fig. 3 shows the pharmacokinetic analysis result in Swiss-Webster mouse.20-2b, α 2b- are applied to mouse 413rd, PEG intrones or PEGASYS and the IFN α 2b concentration in elisa assay blood serum sample was passed through through 96 hours.Show Serum eliminates curve.Serum half-life (T_1/2) supersession rate and mean residence time (MRT) be summarized in the form of insertion.

Fig. 4 (A) shows 20-2b ADCC effector functions.It is in the presence of the PBMC of fresh separated that Daudi or Raji is thin Incubated 4 hours together with born of the same parents and 5 μ g/ml 20-2b, 22-2b, v-mab, epratuzumab (e-mab) or h734, it is then quantitative thin Cellular lysis.

Fig. 4 (B) shows 20-2B CDC effector functions.By Daudi cells and 20-2b in the presence of human complement (●), 734-2b (■) or v-mab (zero) serial dilution incubate together.Draw the complement control % (work in test sample Cell number, compared to only by complement processing cell) relative to nM concentration log values curve.Error bars, SD.

Fig. 5 shows consumption enhancings of the 20-2b to the NHL cells from whole blood.By fresh test tube of hepari human blood with Daudi or Ramos mixing and the 20-2b (●) with 0.01,0.1 or 1nM, v-mab (zero), 734-2b (■) or v-mab+734- 2b () is incubated two days together.Shown treatment is evaluated to lymthoma and the shadow of PBLC using flow cytometry Ring.Error bars, SD.

Fig. 6 (A) shows display 20-2b controlling in dissemination Burkitt lymphoma (Daudi) xenograft models Treat the survival curve of effect.Daudi cells were applied into female C.B.17SCID mouse veins at the 0th day.Treatment is by with single 20-2b (●) that subcutaneous dosage provides, 734-2b (■), v-mab (zero), PEGASYSOr salt solution (X) composition.Treat day Number is indicated with arrow.Use Prism software analysis survival curves.In early stage Daudi model, at the 1st day to 10 mouse Group provides 0.7pmol (solid line) or 0.07pmol (dotted line) single dose.

Fig. 6 (B) shows the research similar with Fig. 6 (A), but late in Daudi models.It is small to 10 at the 7th day The group of mouse provides 0.7pmol (solid line), 7pmol (dotted line) or 70pmol (gray line) single dose.

Fig. 7 (A) presents display 20-2b in dissemination Burkitt lymphoma (Raji and NAMALWA) xenograft mould The survival curve of therapeutic efficiency in type.NHL cells were applied into female C.B.17SCID mouse veins at the 0th day.Treatment by 20-2b (●), 734-2b (■), v-mab (zero) or salt solution (X) composition provided with subcutaneous dosage.Treatment number of days is referred to arrow Show.Use Prism software analysis survival curves.Late in Raji models, 10 mouse of angel in the 5th, 7,9,12,14 and 16 Group receive 250pmol dosage.

Fig. 7 (B) shows the research similar with Fig. 7 (A), but in early stage NAMALWA model.The group of 6 mouse In the 1st, 3,5,8, the 10 and 12 day 20-2b or 734-2b for receiving 250pmol dosage or at the 1st, 5,9,13,17,21 and 25 day Receive the v-mab of 3.5nmol dosage.

Fig. 8 (A) AD2-IFN- λ 1 express the schematic diagram of module.Figure individually discloses SEQ ID NO99- by outward appearance order 100.(B) interferon-antibody DNL is shown^TMThe schematic diagram of the construction of module.

Cell surface expression of Fig. 9 antigens in various cell lines.

Figure 10 (A) (E1)-λ 1 and ME-180 cells, (B) (15)-λ 1 and HepG2 cells and (C) (C2) -1 are thin with A375 The binding activity of born of the same parents has strengthened compared to the modules of AD2- λ 1.

Cytotoxic effects of Figure 11 (the E1)-λ 1 for ME-180 cells.

Cytotoxic effects of Figure 12 (the 15)-λ 1 for ME-180 cells.

Figure 13 antivirus actions.(A) HCV-Ab IgG effect enhancings of (the c225)-λ 1 in Huh-7 cells.With shown concentration (c225)-λ 1, (C2)-λ 1 or HCV genotype 1 b Con1 of the 1 dose of processing of rhIFN- λ with expression firefly luciferase are replicated The Huh-7 stable cell lines of son.After 3 days, uciferase activity is measured and by being dropped relative to the activity of untreated cell Low percentage test antivirus action.Using Graph Pad Prism using S-shaped fitting (variable slope) come analyze data.Sample Independent operation is twice in duplicate for product.(B) anti-EMCV effect enhancings of (the 15)-λ 1 in A549 cells.Excited with EMCV Before, make A549 cells warm together with (15)-λ 1, (C2)-λ 1, rhIFN- λ 1 or hMN15-Fab-DDD2 serial dilution Educate.Range estimation cytopathic effect (CPE) measure is carried out, and S-shaped fitting (variable slope) is used using GraphPad Prism Carry out analyze data.Two parts of ground independent operations of sample are twice.

It is described in detail

Definition

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

As used herein, term " and " can be used for referring to "or" and conjunction or extract.I.e., except as otherwise noted, otherwise this two Individual term is interpreted as being equal to "and/or".

" therapeutic agent " is atom, molecule or the compound that can be used for treatment disease.The example of therapeutic agent includes antibody, antibody Fragment, peptide, medicine, toxin, enzyme, nuclease, hormone, immunomodulator, ASON, siRNA (siRNA), chela Mixture, boron compound, sensitising agent, dyestuff and radio isotope.

" diagnosticum " is atom, molecule or the compound that can be used for diagnosing the illness.Useful diagnosticum includes (but unlimited In) radio isotope, dyestuff (such as together with biotin-Streptavidin compound), contrast agent, fluorescent chemicals or point Son, and the reinforcing agent (for example, paramagnetic ion) for magnetic resonance imaging (MRI).

" antibody " refers to that total length is (i.e. naturally occurring or by normal immunoglobulin gene fragment as used herein What recombination method was formed) immunoglobulin molecules (such as IgG antibody), or exempt from immunocompetence (specifically binding) A part for epidemic disease globulin molecule, as antibody fragment." antibody " includes monoclonal antibody, polyclonal antibody, bispecific Antibody, multi-specificity antibody, rodent antibody, chimeric antibody, humanized antibody and human antibodies.

" exposed antibody " is to be not connected to therapeutic agent or the antibody of diagnosticum or its antigen-binding fragment.The Fc of complete exposed antibody Part can provide effector function, such as complement fixation and ADCC (see, for example, Markrides, Pharmacol Rev50：59- 87,1998).Other mechanism of exposed antibody inducing cell death may include Apoptosis.(Vaswani and Hamilton, Ann Allergy Asthma Immunol81：105-119,1998.)

" antibody fragment " is a part for complete antibody, such as F (ab ')₂、F(ab)₂、Fab′、Fab、Fv、sFv、scFv、 DAb etc..Regardless of structure, antibody fragment is combined with the same antigen that full length antibody is identified.For example, antibody fragment include by The isolated fragment of variable region composition, such as " Fv " fragment that is made up of heavy chain with the variable region of light chain or connected gently by peptide linker Chain and the recombinant single chain peptide molecule of weight chain variable district (" scFv albumen ")." single-chain antibody " (being often abbreviated as " scFv ") is by wrapping Containing V_HAnd V_LThe polypeptide chain composition of domain, the domain interact to form antigen binding site.V_HAnd V_LDomain leads to Often connected by the peptide with 1 to 25 amino acid residue.Antibody fragment also includes double-chain antibody, three chain antibodies and single domain antibody (dAb)。

If antibody or the amount of application of immune conjugate preparation or composition as described herein are physiologically significant, It is referred to as applying with " therapeutically effective amount ".It is detectable that if the presence of medicament causes the physiology of acceptor subject to occur Change, then the medicament is physiologically significant.In certain embodiments, if the presence of antibody preparation cause it is anti- Tumor response or sign and the symptom for alleviating morbid state, then the antibody preparation is physiologically significant.Physiologically Obvious action can also be the body fluid and/or cell immune response for causing acceptor subject, cause target cell growth by To suppression or dead.

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

In preferred embodiments, interferon-antibody complex is with DOCK-AND-LOCK^TM(DNL^TM) composite form Formed (see, for example, United States Patent (USP) No.7,521,056,7,527,787,7,534,866,7,550,143 and 7,666,400, its Respective embodiment part is incorporated herein by reference).In general, the technology, which utilizes, occurs in cAMP dependences The dimerization and dockerin domain (DDD) sequence of regulation (R) subunit of protein kinase (PKA) are with being derived from a variety of AKAP albumen Specificity and high-affinity binding interactions (Baillie etc., FEBS between anchoring domain (AD) sequence of any one Letters.2005；579：3264；Wong and Scott, Nat.Rev.Mol.Cell Biol.2004；5：959).DDD and AD peptides Connectable to any protein, peptide or other molecules.Because DDD sequences spontaneously dimerization and are incorporated into AD sequences, institute Stating technology allows to form compound between any selected molecule connectable to DDD or AD sequences.

Although the DNL of standard^TMCompound includes the trimerization that an AD connection molecule is connected to two DDD connection molecules Body, but the change of composite structure allows to form dimer, tripolymer, the tetramer, pentamer, six aggressiveness and other polymers. In some embodiments, DNL^TMCompound can include two or more antibody, antibody fragment or fusion protein, and it is incorporated into Identical antigenic determinant is incorporated into two or more different antigens.DNL^TMCompound can also include it is one or more its His effector, for example, protein, peptide, immunomodulator, cell factor, interleukins, interferon, associated proteins, peptide ligand, Carrier protein, toxin, ribalgilase such as ranpirnase (onconase), inhibition oligonucleotides such as siRNA, antigen or xenogenesis resist Original, polymer such as PEG, enzyme, therapeutic agent, hormone, cytotoxic agent, anti-angiogenic agent, promote apoptosis agent or any other molecule Or aggregation.

PKA is in the combination by second messenger's cAMP and R subunit and the signal transduction pathway of a best research that triggers In play a key effect, it separates (Walsh etc., J.Biol.Chem.1968 from rabbit skeletal muscle first in nineteen sixty-eight； 243：3763).The structure of holoenzyme be made up of two catalytic subunits that inactive form is remained by R subunits (Taylor, J.Biol.Chem.1989；264：8443).It was found that PKA isoenzymes has two kinds of R subunits (RI and RII), and often One type has α and β isoforms (Scott, Pharmacol.Ther.1991；50：123).Therefore, PKA adjusts the four of subunit Kind isoform is RI α, RI β, RII α and RII β.The R subunits are only with stable dimeric forms separation and dimerization Domain has been shown forms (Newlon etc., Nat.Struct.Biol.1999 by RII α preceding 44 n terminal residues；6：222). As discussed below, the similar portions of the amino acid sequence of other regulation subunits are related in dimerization and stop, are each located on Near the N-terminal for adjusting subunit.The combination of cAMP and R subunits causes broad spectrum serine/threonine kinase of active catalytic subunit The release of activity, the subunit stopped by it together with AKAP PKA is divided be positioned at selected substrate (Scott etc., J.Biol.Chem.1990；265；21561).

Since first AKAP be microtubule associated proteins-1B since being characterized within 1984 (Lohmann etc., Proc.Natl.Acad.Sci US A.1984；81：6723), structure is identified in species of the scope from saccharomycete to the mankind More than 50 kinds various AKAP, it is positioned at various Subcellular locations, including plasma membrane, actin cytoskeleton, nucleus, line Plastochondria and endoplasmic reticulum (Wong and Scott, Nat.Rev.Mol.Cell Biol.2004；5：959).AD for PKA AKAP For amphipathic helix (Carr etc., J.Biol.Chem.1991 with 14-18 residue；266：14188).Single AKAP it Between AD amino acid sequence it is extremely different, wherein report for RII dimers binding affinity scope be 2 to 90nM (Alto etc., Proc.Natl.Acad.Sci.USA.2003；100：4445).AKAP will be only in conjunction with dimerization R subunits.For people Class RII α, AD are incorporated into hydrophobic surface (Colledge and Scott, the Trends Cell formed by 23 n terminal residues Biol.1999；6：216).Therefore, mankind RII α dimerization domain and AKAP binding domain are all positioned at identical N-terminal 44 (Newlon etc., Nat.Struct.Biol.1999 in the sequence of amino acid；6：222；Newlon etc., EMBO are J.2001；20： 1651), it is referred to herein as DDD.

We have developed following platform technology, wherein by the use of the mankind PKA regulation subunit DDD and AKAP AD as A pair of outstanding joint modules, turn into non-covalent complex for stopping any two entity (hereinafter referred to as A and B), it can lead to The strategic location crossed in DDD and AD introduces cysteine residues and further locked as DNL with promoting disulfide formation^TMIt is multiple Compound.The conventional method opinion of methods described is as follows.Entity A is built by DDD sequences to be connected to A precursor, is derived from One part, hereinafter referred to as a.Because the DDD sequences will realize spontaneously forming for dimer, therefore A will be by a₂Group Into.Entity B is built by the way that AD sequences to be connected to B precursor, is derived from second part, referred to as b.a₂In Contained DDD dimerization motif will produce the docking site for being used for being combined with the AD sequences included in b, so as to promote a₂With b's Preparation is associated to be formed by a₂The trimerization compound of the binary of b compositions.The binding events are due to then covalent by disulfide bridge Ground fixes the reaction of two entities and becomes irreversible, because initial binding interactions should make the reaction on DDD and AD Property mercapto locus specificity ground close (Chmura etc., Proc.Natl.Acad.Sci.USA.2001；98：8480) to connect, According to the principle of validity local concentration, the reaction will efficiently occur.Using the various combinations of joint, aptamer module and precursor, It can produce and using extensive a variety of DNL with different stoichiometries^TMConstruct (see, for example, United States Patent (USP) No.7,550, 143rd, 7,521,056,7,534,866,7,527,787 and 7,666,400).

By the way that DDD is connected with functional groups of the AD away from two kinds of precursors, the connection of these locus specificities is it is also contemplated that protect Stay the original activity of both precursors.This method is essentially modular and potentially useful in locus specificity and covalent Ground connects broad range of material, including peptide, protein, antibody, antibody fragment and with broad range of other active effects Answer subdivision., can be by substantially using the fusion protein method of the conjugated effectors of structure AD and DDD described in Examples below Any protein or peptide are incorporated to DNL^TMIn construct.However, the technology is unrestricted and can utilize other conjugated methods.

Become known for manufacturing a variety of methods of fusion protein, including nucleic acid synthesis, hybridization and/or amplification to produce coding institute The synthesis double-strandednucleic acid of fusion protein interested.Such double-strandednucleic acid can be inserted into expression vector, with for use by standard molecule Biology techniques produce fusion protein (see, for example, Sambrook etc., Molecular Cloning, A laboratory Manual, second edition, 1989).In these preferred embodiments, AD and/or DDD can be partially attached to effect protein or The N-terminal or C-terminal of peptide.However, it would be recognized by those skilled in the art that chemical property and effector according to effect subdivision It is related to the part of its physiologically active in part, the site that AD or DDD parts are connected in effect subdivision may be different.It can be used Techniques known in the art carrys out locus specificity and connects a variety of effect subdivisions, for example, using bivalent cross-linking reagent and/or other The conjugated technology of chemistry.

Structure-function relationship in AD and DDD parts

For different types of DNL^TMConstruct, using different AD or DDD sequences.Be provided below exemplary DDD and AD sequences.

DDD1

SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO：1)

DDD2

CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO：2)

AD1

QIEYLAKQIVDNAIQQA(SEQ ID NO：3)

AD2

CGQIEYLAKQIVDNAIQQAGC(SEQ ID NO：4)

It would be recognized by those skilled in the art that DDD1 and DDD2 are the DDD of mankind's RII α isoforms based on protein kinase A Sequence.However, in alternative embodiments, DDD and AD parts are potentially based on the DDD sequences of the protein kinase A of mankind's RI alpha forms Row and corresponding AKAP sequences, illustrated in following article 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, the other sequences variant of AD and/or DDD parts can be used for DNL^TMCompound Structure.For example, only four kinds of variants of mankind's PKA DDD sequences be present, corresponding to PKA RI α, RII α, RI β and RII β DDD portions Point.RII α DDD sequences are above-disclosed DDD1 and DDD2 basis.Four kinds of mankind's PKA DDD sequences are shown below.DDD sequences Row represent RII α residue 1-44, RII β residue 1-44, RI α residue 12-61 and RI β residue 13-66.(notice From mankind PKA RII α DDD parts, slight modification obtains DDD1 sequence.)

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 domains is the theme of research.(see, for example, 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 Deng 2006, Biochem J400：493-99；Gold etc., 2006, Mol Cell24：383-95；Kinderman etc., 2006, Mol Cell24：397-408, its respective full content are incorporated herein by reference.)

For example, (2006, the Mol Cell24 such as Kinderman：It 397-408) have studied the crystalline substance of AD-DDD binding interactions Body structure is simultaneously inferred to mankind's DDD sequences to contain multiple important conserved amino acids in dimer is formed or AKAP is combined residual Base, hereafter SEQ ID NO：It is following in 1 to line out.(referring to Kinderman etc., 2006 Fig. 1, it is herein incorporated by reference Herein.) it would be recognized by those skilled in the art that when designing the sequence variants of DDD sequences, people can it is expected to avoid changing to appoint What residue with underscore, and can be to making conserved amino acid substitution with reference to hardly important residue for dimerization and AKAP.

SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO：1)

Following article is discussed more fully, and 20 kinds of common L-amino acids have each been characterized with conserved amino acid substitution.Therefore, Substituted according to Kinderman (2006) data and conserved amino acid, based on SEQ ID NO：1 potential optional DDD sequences It is shown in Table 1.In the table 1 of design, highly conserved 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor is only accounted for.Have for example, only being substituted with charged residues There is the residue of identical charges, substitute the residue with small side chain with similarly sized residue, only substituted with other hydroxyls Hydroxyl side chains etc..Because proline is not substituted for the unique effect of amino acid secondary structure, proline by other residues.It is limited These potential optional DDD partial sequences of number are shown in hereafter SEQ ID NO：12 to SEQ ID NO：In 31.This area skill Art personnel are it will be recognized that standard technique can be utilized, such as use commercially available peptide synthesizer or well-known direct mutagenesis skill Art builds in DDD partial categories the almost optional material of unrestricted number.Can also be easy by standard binding assay Ground determines the effect that 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor combines for AD parts, such as such as Alto (2003, Proc Natl Acad Sci USA100：Disclosed in 4445-50).

Table 1.DDD1 (SEQ ID NO：1) the conserved amino acid substitution in.Consensus 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)

(2003, the Proc Natl Acad Sci USA100 such as Alto：4445-50) to the AD sequences of various AKAP albumen Progress bioinformatic analysis is referred to as AKAP-IS RII selectivity AD sequences (SEQ ID NO to design：3), wherein DDD Binding constant is 0.4nM.AKAP-IS sequences are designed to combine in PKA AKAP peptide antagonists.Hereafter in SEQ ID NO： Marked in 3 with underscore and tended to reduce the substituted residue combined with DDD in AKAP-IS sequences.Those skilled in the art It will be recognized that when designing the sequence variants of AD sequences, people can it is expected to avoid changing any residue with underscore, and can be right For DDD conserved amino acid substitution is made with reference to hardly important residue.Table 2 shows AKAP-IS sequences (AD1, SEQ ID NO：3) in potential conserved amino acid substitution, with table 1 above on DDD1 (SEQ ID NO：1) it is similar shown in.

Hereafter SEQ ID NO：32 to SEQ ID NO：49 show these a limited number of potential optional AD partial orders Row.In addition, those skilled in the art can be based on the data manufacture of (2003) such as Alto, test and use possible AD partial sequences The material of suitable big figure in classification.It is noted that Alto (2003) Fig. 2 show can be based on actual Binding experiment manufacture and While keep with the binding activity of DDD parts or even very big figure potential 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.

AKAP-IS

QIEYLAKQIVDNAIQQA(SEQ ID NO：3)

Table 2.AD1 (SEQ ID NO：3) the conserved amino acid substitution in.Consensus 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)

(2006, the Mol Cell24 such as Gold：383-95) SuperAKAP-IS sequences are developed using crystallography and peptide screening Arrange (SEQ ID NO：50) PKA RII isoforms five orders of magnitude high compared to the selectivity of RI isoforms, are showed.Not plus under The residue of line indicates that 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor relative to the position of AKAP-IS sequences, which increases the knot with RII α DDD parts Close.In this sequence, N-terminal Q residue is numbered as residue numbering 4, and C-terminal A residues are residue numbering 20.Can be substituted with Influence is residue 8,11,15,16,18,19 and 20 (Gold etc., 2006) for the residue of RII α compatibility.It is it is contemplated that some In optional embodiment, with SuperAKAP-IS sequences AKAP-IS AD partial sequences can be substituted to prepare DNL^TMConstruct. Other the optional sequences that may replace AKAP-IS AD sequences are shown in SEQ ID NO：In 51-53.Relative to AKAP-IS sequences Substitution marks through underscore.It is expected that with SEQ ID NO：AD2 sequences shown in 4 are the same, and AD parts may also include it His N-terminal residue cysteine and glycine and C-terminal residue glycine and cysteine.

SuperAKAP-IS

QIEYVAKQIVDYAIHQA(SEQ ID NO：50)

Optional AKAP sequences

QIEYKAKQIVDHAIHQA(SEQ ID NO：51)

QIEYHAKQIVDHAIHQA(SEQ ID NO：52)

QIEYVAKQIVDHAIHQA(SEQ ID NO：53)

Gold etc. Fig. 2 discloses other DDD binding sequences from a variety of AKAP albumen, is shown in hereafter.

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)

(2006, the Biochem J400 such as Stokka：The peptide competitor for the AKAP for being incorporated into PKA 493-99) is also developed, It is shown in SEQ ID NO：In 64-66.Peptide antagonists are appointed as Ht31 (SEQ ID NO：64)、RIAD(SEQ ID NO：65) and PV-38(SEQ ID NO：66).The compatibility that Ht-31 peptides show for PKA RII isoforms is bigger, and RIAD and PV-38 show Show higher to RI compatibility.

Ht31

DLIEEAASRIVDAVIEQVKAAGAY(SEQ ID NO：64)

RIAD

LEQYANQLADQIIKEATE(SEQ ID NO：65)

PV-38

FEELAWKLAKMIWSDVFQQC(SEQ ID NO：66)

(2006, the Biochem J396 such as Hundsrucker：297-306) develop other peptides for the AKAP for being incorporated into PKA Competitor, for the DDD of PKA RII forms binding constant as little as 0.4nM.The sequence of various AKAP antagonistic peptides is provided in In Hundsrucker etc. table 1, hereafter it is reproduced in table 3.AKAPIS represents synthesis RII subunit binding peptides.Every other peptide is all RII binding domain from shown AKAP.

Table 3.AKAP peptide sequences

Hereafter by for AKAP IS sequences (SEQ ID NO：3) underline to indicate in different AKAP albumen Highly conserved residue in AD domains.The residue is as observed by Alto etc. (2003), wherein with the addition of the ammonia of C-terminal third Sour residue.(referring to Fig. 4 of Hundsrucker etc. (2006), it is incorporated herein by reference.) for RII DDD sequences The sequence of peptide antagonists with special high-affinity be AKAP-IS, AKAP7 δ-wt-pep, AKAP7 δ-L304T-pep and AKAP7 δ-L308D-pep sequence.

AKAP-IS

QIEYLAKQIVDNAIQQA(SEQ ID NO：3)

(2001, the J Biol Chem276 such as Carr：17332-38) it have studied from the mankind and otherwise non-human proteins not Sequence homology degree between same AKAP combination DDD sequences, and identify and seem topnotch in different DDD parts Residue in conservative DDD sequences.These are below by SEQ ID NO：Under 1 mankind's PKA RII α DDD sequences add Rule to indicate.Especially conservative residue is further indicated by italic.The residue and Kinderman etc. (2006) proposition pair It is overlapping but differ in being bound to those residues important for AKAP albumen.It would be recognized by those skilled in the art that designing During DDD sequence variants, most preferably avoid changing most conservative residue (italicization), and further preferably avoid changing guarding Residue (underlines), and is contemplated that and carries out conserved amino acid substitution for the residue for both not underlining non-italic.

SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO：1)

DDD1 (the SEQ ID NO of data based on Carr etc. (2001)：1) conserved amino of one group of sequence by modification Acid substitution is shown in Table 4.Even if the substituted sequence reduced by this group, still suffers from 65, more than 000 kind can be by art technology The possible optional DDD partial sequences that personnel produce, test and used under without excessive experiment.Those skilled in the art can hold Change places to obtain these optional DDD amino acid sequences as disclosed in Tables 1 and 2 above.

Table 4.DDD1 (SEQ ID NO：1) the conserved amino acid substitution in.Consensus is disclosed as SEQ ID NO：89.

It would be recognized by those skilled in the art that using the standard technique in this area and only by normal experiment, in DDD or These and other 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors in AD amino acid sequences can be used for producing the optional material in AD or DDD partial categories.

Interferon and other immunomodulators

In certain preferred aspects, effect subdivision is immunomodulator.Immunomodulator is worked as one kind to be present When can change, suppress or stimulate body immune system agent.The immunomodulator used may include cell factor, stem cell life The long factor, lymphotoxin, hematopoietic factor, colony stimulating factor (CSF), interferon (IFN), hematopoietin, TPO and its combination.It is especially useful that lymphotoxin such as TNF (TNF), hematopoietic factor such as Interleukins (IL), colony stimulating factor such as granulocyte colony stimulating factor (G-CSF) or granular leukocyte macrophage colony thorn Swash the factor (GM-CSF), interferon such as interferon-' alpha ', interferon-beta, interferon-γ or interferon-λ, and stem cell growth because Son is as being referred to as " the S1 factors " person.

In a more preferred embodiment, effect subdivision is cell factor, such as lymphokine, monokine, growth The factor and traditional polypeptide hormone.Cell factor includes growth hormone such as human growth hormone, N- methionyl human growth hormones And bovine growth hormone；Parathyroid hormone；Thyroxine；Insulin；Proinsulin；Relaxain；Relaxation precipitinogen；Glycoprotein hormones Such as follicular stimulating hormone (FSH), thyrotropic hormone (TSH) and interstitialcellstimulating hormone (ICSH) (LH)；Placenta growth factor (PIGF), liver life The long factor；Prostaglandin, fibroblast growth factor；Prolactin(PRL；Human placental lactogen, OB albumen；Tumor necrosis factor-alpha With TNF-β；Mullerian inhibiting substance；Small mouse promoting sexual gland hormone related peptide；Inhibin；Activin；Vascular endothelial growth The factor；Integrin；TPO (TPO)；Nerve growth factor such as NGF- β；Platelet growth factor；TGF (TGF) such as TGF- α and TGF-β；Insulin like growth factor-1 and Insulin-like growth factor-II；Hematopoietin (EPO)；Bone-inducing factor；Interferon such as interferon-' alpha ', interferon-beta, interferon-λ and interferon-λ；Colony stimulating factor (CSF) such as macrophage-CSF (M-CSF)；Interleukins (IL) such 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, TNF (TNF, such as TNF-α) and LT.In an especially preferred embodiment, cell factor is IFN-α 2b.

Protein or the amino acid sequence of peptide immunomodulator such as cell factor are well-known in the art, and can be Any such known array is used in the practice of the present invention.Skilled in the art realises that the public information of cytokine sequence A variety of sources.For example, ncbi database includes the protein and nucleic acid sequence encoding of large amount of cell factor and immunomodulator, Such as hematopoietin (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 substantially any peptide or protein matter immunological regulation listed above Agent.The appropriate amino acid and/or nucleotide sequence for identifying substantially any interested protein or peptide effect subdivision are these The customary affairs of art personnel.The commercial source of cell factor can also be obtained and can be used, such as total length mankind's IFN-α 2b cDNA clones (the human cloned catalogue #HORF01 clone ID IOH35221 of Invitrogen Ultimate ORF).

Antibody

In certain embodiments, antibody or its antigen-binding fragment may be incorporated into DNL^TMIn construct, such as by that will resist Body or fragment are connected to interferon or other cell factors for cell factor targeted delivery.Any of antibody or its antigen Binding fragment may be incorporated into DNL^TMIn construct.In preferred embodiments, compound is used for cancer therapy and antibody knot Together in tumor associated antigen (TAA).Kinds of tumors related antigen is as known in the art, including but 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, fiber connection ED-B, factor H, FHL-1, Flt-3, folacin receptor, GROB, HMGB-1, hypoxia inducible factor (HIF), HM1.24, the pancreas of albumen Island element like growth factor -1 (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 antigens, tumor necrosis antigens, TNF-α, TRAIL acceptors (R1 and R2), VEGFR, EGFR, PlGF, complement because Sub- C3, C3a, C3b, C5a, C5 and oncogene products.Other kinds of target antigen can be used for various disease state based on The therapy of antibody and the available DNL for being associated with the antibody for targetting any this kind of optional antigen^TMConstruct.

Available for DNL^TMExemplary anticancrin in construct includes but is not limited to hR1, and (anti-IGF-1R, the U.S. are interim Patent Application Serial No.61/145,896, be filed in 1/20/09), hPAM4 (anti-MUC1, United States Patent (USP) No.7,282,567), HA20 (anti-CD20, United States Patent (USP) No.7,251,164), hA19 (anti-CD19, United States Patent (USP) No.7,109,304), hIMMU-31 (anti-AFP, United States Patent (USP) No.7,300,655), hLL1 (anti-CD74, United States Patent (USP) No.7,312,318), hLL2 (anti-CD22, it is beautiful State patent No.7,074,403), hMu-9 (anti-CSAp, United States Patent (USP) No.7,387,773), (anti-HLA-DR, the U.S. are special by hL243 Sharp No.7,612,180), hMN-14 (anti-CEACAM5, United States Patent (USP) No.6,676,924), hMN-15 (anti-CEACAM6, the U.S. Patent application No.10/672,278), (anti-CEA, the U.S. are special by hRS7 (anti-EGP-1, United States Patent (USP) No.7,238,785) and hMN-3 Sharp No.7,541,440), the embodiment part of each cited patent or application is incorporated herein by reference.This area It will be recognized that this list is without limitation and any other known anti-TAA antibody can be incorporated into DNL^TMStructure Build in body.

Antigen binding antibody fragment is well known in the art, such as F (ab ')₂、F(ab)₂、Fab′、Fab、Fv、 ScFv etc., and any such known fragment can be used.As used herein, antigen binding antibody fragment refer to combine can be complete Any fragment of the antibody of the same antigen of whole or parental antibody identification.Become known for preparing substantially any interested resist The technology of body or AD the and/or DDD conjugates of fragment (for example, United States Patent (USP) No.7,527,787).

It is workable not conjugated to be referred to as " naked " antibody or its fragment in the antibody of therapeutic agent or its fragment.Optional real Apply in scheme, can be by antibody or fragment conjugate together in one or more therapeutic agents and/or diagnosticum.It is known in the art more extensively The such therapeutic agent of kind and diagnosticum, following article are discussed more fully, and any such known therapeutic agent or diagnosticum can be used.

Technology for preparing the anti-substantially monoclonal antibody of any target antigen is well known in the art.Referring to Such as Kohler and Milstein, Nature256：495(1975)；And the (eds.) such as Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, volume 1, the 2.5.1-2.6.7 pages (John Wiley＆Sons 1991).Briefly, can be by following Obtain monoclonal antibody：Into mouse, injection includes the composition of antigen, removes spleen to obtain bone-marrow-derived lymphocyte, and B lymphs is thin Born of the same parents are merged with myeloma cell produces hybridoma, clones the hybridoma, and selection produces the positive colony of the antibody for antigen, The clone of the antibody for producing and being directed to antigen is cultivated, and the antibody is separated from Hybridoma culture.

MAb from Hybridoma culture can be separated and purified by a variety of perfect technologies.Such isolation technics includes Use the affinity chromatography, SEC and ion-exchange chromatography of Protein A sepharose.See, for example, Coligan's The 2.7.1-2.7.12 pages and the 2.9.1-2.9.3 pages.In addition, referring to Baines etc., " Purification OfImmunoglobulin G (IgG), " METHODS IN MOLECULAR BIOLOGY, volume 10, the 79-104 pages (The Humana Press, Inc.1992).

After antibody of the initial generation for immunogene, antibody can be sequenced and then be prepared by recombinant technique.Ability Field technique personnel are it is known that the humanization of rodent antibody and antibody fragment and chimerization.Using from humanization, chimeric or people The antibody repertoire of antibody-like eliminates the potential problems related to the immunogenicity of muroid constant region.

Chimeric antibody

Chimeric antibody is a kind of recombinant protein, and wherein the variable region of human antibodies is by the variable region of such as mouse antibodies (complementary determining region (CDR) for including mouse antibodies) is replaced.When being applied to subject, chimeric antibody shows immunogenicity drop The increase of low and stability.The general technology in clone's muroid immunoglobulin variable domain domain is disclosed in such as Orlandi, Proc.Nat′l Acad.Sci.USA86：In 3833 (1989).Those skilled in the art are it is known that the skill of structure chimeric antibody Art.For example, Leung etc., Hybridoma13：In 469 (1994), by that will encode muroid LL2, (a kind of anti-CD22 monoclonals will resist Body) V_κAnd V_HThe DNA sequence dna of domain and corresponding mankind κ and IgG₁Constant region domain combines and produces LL2 chimeras.

Humanized antibody

Technology for producing humanization MAb be it is well known in the art (see, for example, 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)).Can be by will be small The corresponding variable domains that the heavy variable chains of rat immune globulin and the mouse CDR of light variable chains are transferred to human antibodies will be embedding Conjunction or murine monoclonal antibody humanization.Mouse framework region (FR) in chimeric mAb is also by mankind's FR sequence substitutions. The reduction of antibody compatibility is frequently resulted in mankind FR due to being simply transferred to mouse CDR or even is lost, it is thus possible to is needed Extra modification is wanted to recover the original compatibility of rodent antibody.This can be by the way that one or more of FR areas human residue be put It is changed to its muroid homologue and obtains the antibody to its epitope with good combination compatibility and realize.See, for example, Tempest Deng Biotechnology9：266 (1991) and Verhoeyen etc., Science239：1534(1988).Generally, with muroid pair Substitution will be turned into by answering thing different and being located adjacent to or contact the mankind FR amino acid residues of one or more cdr amino acid residues Candidate.

Human antibodies

Complete human antibodies are produced using combined method or the transgenic animals converted with human immunoglobulin gene seat Method be as known in the art (for example, Mancini etc., 2004, New Microbiol.27：315-28；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 can also be by gene or chromosomal transfection methods and thermophilic bacterium Body display technique construction, the technology are all as known in the art.See, for example, McCafferty etc., Nature348：552- 553(1990).Such human antibodies completely are expected to show the side effect less than chimeric or humanized antibody, and play in vivo The function of substantially endogenous human antibodies.In certain embodiments, methods and procedures claimed is available passes through this Human antibodies caused by class technology.

In another case, display technique of bacteriophage can be used to generate human antibodies (for example, Dantas-Barbosa Deng 2005, Genet.Mol.Res.4：126-40).From normal human subject or the mankind of particular disease states such as cancer can be shown Generate human antibodies (Dantas-Barbosa etc., 2005).It is to circulate from the advantages of individual structure human antibodies of illness and resists Body storehouse may have a preference for the antibody of anti-disease related antigen.

In a non-limiting examples of this method, Dantas-Barbosa etc. (2005) is constructed from osteosarcoma The phage display library of mankind's Fab antibody fragment of patient.Generally, total serum IgE obtains (being same as above) by blood circulation lymphocyte. Recombinant Fab is cloned from the antibody library of μ, γ and κ chain and is inserted in phage display library and (is same as above).RNA is converted into eDNA simultaneously For using the specific primer for heavy chain and light chain immunoglobulins sequence produce Fab cDNA libraries (Marks etc., 1991, J.Mol.Biol.222：581-97).According to Andris-Widhopf etc. (2000, in Phage Display In Laboratory Manual, the (eds.) such as Barbas, the 1st edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, the page 9.1 to 9.22) carry out library structure.Using restriction endonuclease digestion most Whole Fab fragments simultaneously insert phage genome to generate phage display library.It can be bitten by standard as known in the art Phage display method come screen the library (see, for example, Pasqualini and Ruoslahti, 1996, Nature380：364- 366；Pasqualini, 1999, The Quart.J.Nuel.Med.43：159-162).

Phage display can be carried out in a variety of formats, and it is summarized see, for example, Johnson and Chiswell, Current Opinion in Structural Biology3：5564-571(1993).Also the mankind can be generated by the B cell of Activated in Vitro Antibody.Referring to United States Patent (USP) No.5,567,610 and 5,229,275, it is incorporated herein by way of being cited in full text.This area It will be recognized that these technologies are exemplary, and resist using any of for preparing and screening the mankind The method of body or antibody fragment.

It is another it is optional in the case of, using standard immunization protocol, using being resisted by genetically engineered with producing the mankind The transgenic animals generation of body resists the antibody of substantially any immunogenicity target.Human antibodies are obtained from transgenic mice Method is disclosed in Green etc., Nature Genet.7：13(1994)；Lonberg etc., Nature368：856(1994)；And Taylor etc., Int.Immun.6：In 579 (1994).One non-limiting examples of such a system are from Abgenix (Fremont, CA's)(for example, Green etc., 1999, J.Immunol.Methods231：11-23).In similar animal, mouse antibody gene has been inactivated and substituted with feature human antibody gene, and The remainder of mouse immune system keeps complete.

Use part (including most of variable region sequences and auxiliary gene and tune containing mankind's IgH and Ig kappa gene seat Control sequence) reproduction series structure YAC (yeast artificial chromosomes) conversion.Human variable region storehouse can use The B cell of antibody is produced in generation, it can be treated as hybridoma by known technology.It is immunized using target antigenHuman antibodies will be produced by normal immunoreaction, it can be gathered by standard technique discussed above And/or prepare.It is a variety ofStrain is available, and each can produce different types of antibody.Transgenosis Caused human antibodies have been shown with treatment potentiality, while keep the pharmacokinetic property (Green of normal human subject antibody Deng 1999).It would be recognized by those skilled in the art that composition claimed and method are not limited to useSystem, and using by genetically engineered to produce any transgenic animals of human antibodies.

Antibody fragment

The antibody fragment of identification specificity epitope can be generated by known technology.Antibody fragment is the antigen-binding portion of antibody Point, such as F (ab ')₂、Fab′、F(ab)₂, Fab, Fv, sFv etc..F(ab′)₂Fragment can by pepsin digested antibody molecule and Produce, and Fab ' fragments can be by reducing F (ab ')₂The disulfide bond of fragment and generate.Alternatively, Fab ' expression libraries can be built (Huse etc., 1989, Science, 246：1274-1281) to allow to being carried out with required specific monoclonal Fab ' fragments Rapid and easy identification.F(ab)₂Fragment can be generated by Papain digestion of antibodies, and Fab fragments can be by reducing two sulphur Key obtains.

Single Chain Fv Molecule A (scFv) includes VL domains and VH domains.VL and VH domains associate to form target bound site Point.The two domains are further covalently attached by peptide linker (L).Prepare scFv molecules and design the side of suitable peptide linker Method is described below：United States Patent (USP) No.4,704,692；United States Patent (USP) No.4,946,778；R.Raag and M.Whitlow, " Single Chain Fvs. " FASEB volumes 9：73-80(1995)；And R.E.Bird and B.W.Walker, " Single Chain Antibody Variable Regions, " TIBTECH, volume 9：132-137(1991).

Technology for producing single domain antibody (DAB) is also known in the art, such as by reference simultaneously Enter (2006, the Prot Express Purif51 such as Cossins herein：Disclosed in 253-259).

Can by by full length antibody proteolysis or in Escherichia coli (E.coli) or another host expression encode it is anti- The DNA of body fragment prepares antibody fragment.Pepsin or papain digestion full length antibody can be used by conventional method And obtain antibody fragment.These methods have been described in such as Goldenberg United States Patent (USP) No.4,036,945 and 4,331, 647 and bibliography contained therein in.In addition, referring to Nisonoff etc., Arch Biochem.Biophys.89：230 (1960)；Porter, Biochem.J.73：119(1959)；Edelman etc., METHODS IN ENZYMOLOGY, volume 1, Page 422 (Academic Press1967)；And the 2.8.1-2.8.10 pages of Coligan and the 2.10.-2.10.4 pages.

Known antibodies

Can be from the commercially available antibody used in a variety of known sources extensively.For example, by American type culture collection (ATCC, Manassas, VA) can obtain Multiple Antibodies secretory hybridoma cell line.Largely for various disease targets (bag Include but be not limited to tumor associated antigen) antibody in ATCC preservations and/or variable region sequences have been disclosed and can be used for institute In claimed method and composition.See, for example, United States Patent (USP) 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 It is exemplary and be known in the art extensive various other antibody and its hybridoma.It would be recognized by those skilled in the art that The antibody of interested selected disease associated target can be directed to by simple search in ATCC, NCBI and/or USPTO database And obtain the antibody sequence or antibody secreting hybridoma for substantially any Disease associated antigens.Use many institute's weeks in this area The standard technique known, the antigen-binding domains of clonal antibody can be expanded, shearing, connection enters expression vector, and transfection enters Adapt to host cell and for producing protein.

Specific antibodies available for the cancer therapy in the range of method and composition claimed include (but unlimited In) LL1 (anti-CD74), epratuzumab (LL2) and RFB4 (anti-CD22), (anti-Glycoproteins in Epithelial -1 (EGP-1) is anti-by RS7 TROP-2), (anti-carcinoembryonic antigen (CEACAM5, also referred to as CD66e), Mu-9 are (anti-by PAM4 and KC4 (being anti-stick albumen), MN-14 Colon-specific antigen-p), Immu-31 (Anti-α-Fetoprotein), anti-TAG-72 (such as CC49), anti-Tn, J591 or HuJ591 (anti-PSMA (PSMA)), AB-PG1-XG1-026 (anti-PSMA dimers), 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 is (anti- CD20)；Tositumomab (anti-CD20)；GA101 (anti-CD20)；Trastuzumab (anti-CD20) is tieed up, and Herceptin (resists ErbB2).These antibody be it is as known in the art (for example, United States Patent (USP) No.5,686,072,5,874,540,6,107, 090、6,183,744、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 (abstention), 20050271671 and 20060193865；The embodiment part of each document is incorporated herein by reference).What is used is specific Known antibodies include hPAM4 (United States Patent (USP) No.7,282,567), hA20 (United States Patent (USP) No.7,251,164), the hA19 (U.S. Patent No.7,109,304), hIMMU31 (United States Patent (USP) No.7,300,655), hLL1 (United States Patent (USP) No.7,312,318), HLL2 (United States Patent (USP) No.7,074,403), hMu-9 (United States Patent (USP) No.7,387,773), hL243 (United States Patent (USP) No.7, 612,180), hMN-14 (United States Patent (USP) No.6,676,924), hMN-15 (United States Patent (USP) No.7,541,440), (U.S. is special by hR1 Profit application 12/772,645), hRS7 (United States Patent (USP) No.7,238,785), hMN-3 (United States Patent (USP) No.7,541,440), AB- PG1-XG1-026 (U.S. Patent application 11/983,372, being deposited with ATCC PTA-4405 and PTA-4406) and D2/B (WO2009/130575), each patent or the full text of application on drawings and examples part in a manner of being quoted It is incorporated herein.

Anti-TNF-α antibody is as known in the art and can be used for treating immunological diseases, such as asthma.It is known to be directed to The antibody of TNF-α include human antibodies CDP571 (Ofei etc., 2011, Diabetes45：881-85)；Rodent antibody MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI, M302B and M303 (Thermo Scientific, Rockford, IL)；English profit former times is single Anti- (Centocor, Malvern, PA)；Pegylation match trastuzumab (UCB, Brussels, Belgium)；And A Damu Monoclonal antibody (Abbott, Abbott Park, IL).These and many other known anti-TNF-α antibodies are available for claimed In method and composition.Other antibody used include but is not limited to anti-B cell antibody, such as tie up trastuzumab, according to pa pearl Monoclonal antibody, meter La Zhu monoclonal antibodies or hL243；Torr pearl monoclonal antibody (anti-IL-6 acceptors)；Basiliximab (anti-CD25)；Daclizumab is (anti- CD25)；Efalizumab (anti-CD11a)；Muromonab-CD3 (AntiCD3 McAb acceptor)；Anti-CD 40 L (UCB, Brussels, Belgium)；Natalizumab (integrin of anti alpha 4) and omalizumab (anti-IgE).

Macrophage migration inhibitory factor (MIF) be congenital and adaptive immunity and Apoptosis important regulation because Son.It has been reported that CD74 is MIF endogenous recipient (Leng etc., 2003, J Exp Med197：1467-76).Antagonism resists Therapeutic action of the CD74 antibody for the MIF intracellular pathways mediated can be used for treating extensive various disease states, such as bladder Cancer, prostate cancer, breast cancer, lung cancer, colon cancer and chronic lymphocytic leukemia (for example, Meyer-Siegler etc., 2004, BMC Cancer12：34；Shachar and Haran, 2011, Leuk Lymphoma52：1446-54)；Nephrosis, such as kidney Dirty allograft rejection reacts (Lan, 2008, Nephron Exp Nephrol.109：e79-83)；And numerous hair (Meyer-Siegler etc., 2009, Mediators Inflamm, electronic publishing was on March 22nd, 2009 for inflammatory disease； Takahashi etc., 2009, Respir Res10：33).Meter La Zhu monoclonal antibodies (hLL1) are that have to be used to treat the mediated diseases of MIF Therapeutical uses exemplary anti-CD74 antibody.

The pharmaceutical composition of the present invention can be used for treatment tested with neurodegenerative disorders such as Alzheimer's Person.A bar pearl monoclonal antibody is in the clinical test for the treatment of of alzheimer's disease.It is proposed to be used in treatment of alzheimer's disease Other antibody include Alz50 (Ksiezak-Reding etc., 1987, J Biol Chem263：7943-47), more spit of fland reed monoclonal antibody With Su Lan pearl monoclonal antibodies.Infliximab (a kind of anti-TNF-α antibody) has been reported and can reduce amyloid patch and raising Cognition.Because CD74 expresses (Bryan etc., Mol in the neurofibrillary tangles of Alzheimer patients' brain Neurodegeneration2008；3：13), therefore CD74 is by anti-the CD74 antagonistic peptides or antibody of exclusive use or passed through As described herein DNL constructs are in the form of conjugates using treating another target of this disease.

Other workable antibody include being directed to infectious diseases pathogen such as bacterium, virus, mycoplasma or other cause of diseases The antibody of body.Many is as known in the art and any this known antibody for the antibody of these infectious pathogens It can be used in method and composition claimed.For example, the gp120 sugar for human immunodeficiency virus I (HIV-1) The antibody of proteantigen is known, and some this antibody can have immanoprotection action in the mankind.See, for example, Rossi etc., Proc.Natl.Acad.Sci.USA.86：8055-8058,1990.Known ANTI-HIV DRUGS include by (AIDS.2006 October 3 such as Johansson；20(15)：1911-5) the anti-anti-envelope antibodies of description, and by Polymun (Vienna, Austria) is described and the ANTI-HIV DRUGS of sale, and 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, all it is incorporated herein by reference.

Zygoblast, merozoite, schizont and gametophyte stage can be directed to for the antibody of malarial parasite.Pin is generated To the monoclonal antibody of zygoblast (Circumsporozoite antigen), and shown its in vitro with zygoblast can be neutralized in rodent (N.Yoshida etc., Science207：71-73,1980).Some team have developed toxoplasma antibody, and Infection of Toxoplasma Gondii is arch Involved protozoon parasite (Kasper etc., J.Immunol.129 in parasitosis：1694-1699,1982；Ibid, 30： 2407-2412,1983).The antibody for virgin worm surface antigen is developed and has had found that it is acted in vivo or in vitro 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 is sucked blood.Generate in body It is outer specificity suppress parasite a kind of form to another form (epimastigote to trypomastigote stage) differentiation and with The antibody of cell surface glycoprotein reaction；However, this antigen (Sher is not present in mammal (blood flow) form of parasite Deng, Nature, 300：639-640,1982).

Anti fungal antibody is as known in the art, such as anti-sclerotiniose antibody (United States Patent (USP) 7,910,702)；Anti- grape Uronic acid xylose mannosan antibody (Zhong and Priofski, 1998, Clin Diag Lab Immunol5：58-64)；It is anti- Mycotoruloides antibody (Matthews and Burnie, 2001,2：472-76)；And anti-glycosyl sphingolipid antibody (Toledo etc., 2010, BMC Microbiol10：47).

It is (bacterium, virus, primary dynamic the microorganism for causing largely to infect in the mankind for most of has been developed Thing, fungi, other parasites) suitable antibodies, and many be previously used for in-vitro diagnosis purpose.It can be given birth to by conventional method Into these antibody and modern antibody suitable for the present invention.

Antibody is special-shaped

The immunogenicity of therapeutic antibodies increases to infusion reaction risk and the reduction of therapeutic response duration is related (Baert etc., 2003, N Engl J Med348：602-08).Therapeutic antibodies can be determined by the profiled portion of antibody to exist Degree (Stickler etc., 2011, Genes and Immunity12 of immune response are induced in host：213-21).Antibody is different Type is relevant with the amino acid sequence change of ad-hoc location in the constant-region sequences of antibody.IgG comprising heavy chain γ type constant regions resists The abnormal shape of body is referred to as Gm abnormal shapes (1976, J Immunol117：1056-59).

For common IgG1 human antibodies, most common abnormal shape is G1m1 (Stickler etc., 2011, Genes and Immunity12：213-21).However, it is special-shaped (being same as above) that G1m3 is also frequently occurred in white people.It has been reported that G1m1 antibody Containing special-shaped sequence, when being applied to non-G1m1 (nG1m1) acceptor such as G1m3 patient, the special-shaped sequence is tended to induction and exempted from Epidemic disease reacts (being same as above).When being applied to G1m1 patient, it is in immunogenicity (being same as above) that non-G1m1 haterotypic antibodies, which are not,.

Mankind G1m1 abnormal shapes in heavy chain IgG1 CH3 sequences included in Kabat positions 356 amino acid aspartic acid and Leucine in Kabat positions 358.NG1m1 is special-shaped included in the amino acids glutamic acid of Kabat positions 356 and in Kabat positions 358 methionine.The special-shaped glutaminic acid residue for being included in Kabat positions 357 of G1m1 and nG1m1 and it is described it is special-shaped sometimes by Referred to as DEL and EEM is special-shaped.Show exemplary antibodies Rituximab (SEQ ID NO：And dimension trastuzumab (SEQ ID 85) NO：86) non-limiting examples of the heavy chain constant region sequence of G1m1 and nG1m1 haterotypic antibodies.

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

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGG

PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY

NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK

SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Tie up trastuzumab weight chain variable district (SEQ ID NO：86

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG

PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY

NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK

SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Jefferis and Lefranc (2009, mAb1：1-7) commented the special-shaped characteristic sequence changes of IgG and its for The influence of immunogenicity.They report the special-shaped arginine residues for being characterised by Kabat positions 214 of G1m3, different with G1m17 Kabat214 lysine residue is contrasted in type.NG1m1, the 2 special-shaped glutamic acid for being characterised by Kabat positions 356, The methionine of Kabat positions 358 and the alanine of Kabat positions 431.G1m1,2 abnormal shapes are characterised by Kabat positions 356 Aspartic acid, the leucine of Kabat positions 358 and the glycine of Kabat positions 431.Except heavy chain constant region sequence variant it Outside, Jefferis and Lefranc (2009) also reported κ constant region of light chain allotypic variants, and wherein Km1 abnormal shapes are characterised by The valine of Kabat positions 153 and the leucine of Kabat positions 191, Km1,2 abnormal shapes are characterised by Kabat positions 153 Alanine and the leucine of Kabat positions 191, and the special-shaped alanine and Kabat for being characterised by Kabat positions 153 of Km3 The valine of position 191.

On therapeutic antibodies, trastuzumab and Rituximab are tieed up respectively by humanization and for the chimeric of CD20 IgG1 antibody is used for the treatment of extensive a variety of hematology's malignant tumours.Table 5 compares Rituximab with tieing up trastuzumab Special-shaped sequence.As shown in table 5, Rituximab (G1m17,1) is DEL abnormal shape IgG1, wherein in the (heavy chain of Kabat positions 214 CH1 other sequence variation) is lysine in Rituximab, relative to being arginine in trastuzumab is tieed up.Report Immunogenicity of the road dimension trastuzumab in subject less than Rituximab (see, for example, Morchhauser etc., 2009, J Clin Oncol27：3346-53；Goldenberg etc., 2009, Blood113：1062-70；Robak and Robak, 2011, BioDrugs25：13-25), this effect is attributed to the difference between humanized antibody and chimeric antibody.However, EEM and The immunogenicity that special-shaped difference between DEL abnormal shapes may also result in dimension trastuzumab is relatively low.

Abnormal shape of the Rituximab of table 5. to dimension trastuzumab

In order to reduce immunogenicity of the therapeutic antibodies in nG1m1 genotype individuals, it is desirable to which selection corresponds to following Antibody is special-shaped：G1m3 is special-shaped, is characterised by Kabat214 arginine；And nG1m1,2 is empty special-shaped, is characterised by Kabat positions The alanine of 356 glutamic acid, the methionine of Kabat positions 358 and Kabat positions 431.Surprisingly it was found that Repetition subcutaneous administration of the G1m3 antibody within a very long time does not produce significant immune response.In optional embodiment In, the heavy chain of IgG 4 as G1m3 abnormal shapes has Kabat214 arginine, Kabat356 glutamic acid, Kabat359 Methionine and Kabat431 alanine.Because immunogenicity seems at least residue with those positions to a certain extent It is relevant, so it is also a preferred embodiment that the heavy chain constant region sequence of IgG 4 is used for into therapeutic antibodies. The combination of G1m3IgG1 antibody and IgG4 antibody can also be used for therapeutic administration.

49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor

In certain embodiments, disclosed method and composition may relate to the amino with one or more substitutions The generation and use of the protein or peptide of sour residue.In a non-limiting examples, for manufacturing DNL^TMThe DDD of construct And/or AD sequences can be further optimized, such as to increase DDD-AD binding affinities.

It will be recognized by those skilled in the art generally, 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor is typically related to a kind of amino acid With another amino acid replacement (that is, conserved amino acid substitutes) with relatively similar characteristic.The characteristic and ammonia of various amino acid Influence of the base acid substitution to protein structure and function is furtherd investigate and is known in the art.

Such as, it is contemplated that the hydrophilic index of amino acid (Kyte and Doolittle, 1982, J.Mol.Biol., 157：105- 132).The relative hydropathic character of amino acid facilitate gained Secondary structure, its in turn again define the protein with The interaction of other molecules.Hydrophobicity and charge characteristic based on amino acid assign a hydrophilic index to every kind of amino acid (Kyte and Doolittle, 1982), it is specially：Isoleucine (+4.5)；Valine (+4.2)；Leucine (+3.8)；Phenylpropyl alcohol ammonia Sour (+2.8)；Cysteine/cystine (+2.5)；Methionine (+1.9)；Alanine (+1.8)；Glycine (- 0.4)；Soviet Union's ammonia Sour (- 0.7)；Serine (- 0.8)；Tryptophan (- 0.9)；Tyrosine (- 1.3)；Proline (- 1.6)；Histidine (- 3.2)；Paddy Propylhomoserin (- 3.5)；Glutamine (- 3.5)；Aspartic acid (- 3.5)；Asparagine (- 3.5)；Lysine (- 3.9)；And arginine (-4.5).When carrying out conservative replaces, the preferred amino acid using hydrophilic index in ± 2, more preferably in ± 1, with And even more preferably in ± 0.5.

49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor also contemplates for the hydrophily (for example, United States Patent (USP) No.4,554,101) of amino acid residue.Ammonia Base acid residue is endowed hydrophilicity value：Arginine (+3.0)；Lysine (+3.0)；Aspartic acid (+3.0)；Glutamic acid (+3.0)； Serine (+0.3)；Asparagine (+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).It is excellent Amino acid replacement is carried out from other similar hydrophilic amino acid.

Others consider the size for including amino acid side chain.For example, generally not preferably by the amino acid with close side chain If glycine or Serine are into the amino acid with bulky side chains such as tryptophan or tyrosine.Various amino acid residues are to egg The influence of white matter secondary structure is also a kind of consideration.By positive research, different amino acid residues is for protein domain Take alpha-helix, beta sheet or invert secondary structure propensities influence be determined and be known in the art (see, 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 the consideration and deep positive research, conserved amino acid substitution table is built and has been known in the art.Example Such as：Arginine and lysine；Glutamic acid and aspartic acid；Serine and threonine；Glutamine and asparagine；And figured silk fabrics ammonia Acid, 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。

Whether other include the residue positioned at protein interior or exposed to solvent on the consideration of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.For Internal residues, conservative replacement include：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.(see, for example, rockefeller.edu PROWL Website).For the residue exposed to solvent, conservative replacement includes：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.(being same as above) has built various matrix and has come assisted Selection 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, such as PAM250 scorings Matrix, Dayhoff matrix, Grantham matrix, McLachlan matrix, Doolittle matrix, Henikoff matrix, Miyata Matrix, Fitch matrix, Jones matrix, Rao matrix, Levin matrix and Risler matrix (being same as above).

When determining 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, people also contemplate for intermolecular or intramolecular bond presence, such as positively charged residual The formation of ionic bond (salt bridge) between base (such as His, Arg, Lys) and negatively charged residue (such as Asp, Glu), or it is similar Disulfide bond between cysteine residues.

It is known that with the method for any any other amino acid of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in coded protein sequence, and be The normal experiment affairs of those skilled in the art, such as taken by side-directed mutagenesis or by synthesizing and assembling coded amino acid Simultaneously montage enters expression vector constructs to the oligonucleotides in generation.

It is fit

In certain embodiments, the targeting moiety used can be fit.Structure well known in the art and measure are suitable The method of the binding characteristic of body.For example, such technology is described in United States Patent (USP) No.5,582,981,5,595,877 and 5,637, In 459, its respective embodiment part is incorporated herein by reference.Prepare and screen and be interested to being incorporated into specific The fit method of target is 475, the 096 and United States Patent (USP) No.5 it is well known that such as United States Patent (USP) No.5,270,163, Its respective embodiment part is incorporated herein by reference.

It is fit can by any known method (including synthesize, recombinate and purification process) prepare, and can be used alone or Have specific ligand combination to identical target with other to use.It is generally desirable to a minimum of about 3 nucleotides, preferably at least 5 Individual nucleotides specifically binds to realize.Be shorter than the fit to be feasible of the sequence of 10 bases, but with 10,20,30 or 40 the fit of nucleotides are preferable.

It is fit can through separate, be sequenced and/or expand or in the form of conventional DNA or RNA molecule synthesize.Alternatively, feel emerging The fit oligomer for including modification of interest.In fit generally existing any hydroxyl can phosphonate ester base, phosphate-based displacement, Protected by the protection group of standard, or be activated to prepare the other connection with other nucleotides, or can be conjugated to solid support Thing.One or more phosphodiester bonds can be replaced by optional linking group, such as P (O) O are by P (O) S, P (O) NR₂、P(O)R、 P (O) OR ', CO or CNR₂Displacement, wherein R is H or alkyl (1-20C) and R ' are alkyl (1-20C)；In addition, this group can Adjacent nucleotides is connected to by O or S.All connections in oligomer are without identical.

Affine body and Fynomer

Some optional embodiments can replace antibody using affine body.Affine body is available commercially from Affibody AB (Solna, Sweden).Affine body acts as antibody analog and for combining the little albumen matter in target molecules.By in α spiral shells Carry out integration engineering on rotation protein backbone and produce affine body (Nord etc., 1995, Protein Eng8：601-8；Nord Deng 1997, Nat Biotechnol15：772-77).Affine body design is three spiral shells based on the IgG binding domain comprising a-protein Rotation binding structure (Nord etc., 1995；1997).Involved 13 kinds of amino acid in bacterioprotein A Fc binding activity can be passed through Randomization come produce with a variety of binding affinities affine body (Nord etc., 1995；1997).After randomisation, by PCR Library clone is expanded into phagicin carrier to be screened for use by the phage display of mutain.Standard can be used Phage display triage techniques (for example, Pasqualini and Ruoslahti, 1996, Nature380：364-366； Pasqualini, 1999, Quart.J.Nucl.Med.43：159-162) phage display technology is screened for any of antigen Show library, with the one or more affine bodies for being directed to target antigen of identification.

Have to HER2/neu specific¹⁷⁷The affine body of Lu marks has proven to internal targeted expression HER2 heterograft Thing (Tolmachev etc., 2007, Cancer Res67：2773-82).Although due to low molecule amount radio-labelled compound Renal toxicity caused by accumulation is initially a problem, but reduces kidney accumulation with the Reversible binding of albumin, is achieved with quilt The therapy (being same as above) based on radionuclide that the affine body of mark is carried out.

Recently it has been proved feasibility (the Tolmachev for being used for in-vivo tumour using radiolabeled affine body and being imaged Deng 2011, Bioconjugate Chem22：894-902).The NOTA of maleimide derivative it is conjugated in the affine bodies of anti-HER2 simultaneously With¹¹¹In radioactive labels (are same as above).The mouse of the DU-145 xenograft with expression HER2 is applied to, then carries out γ Camera imaging, it is allowed to the visualization (being same as above) of xenograft.

Fynomer, which can also be incorporated into, has similar compatibility and specific target antigen to antibody.Fynomer is to be based on Mankind's Fyn SH3 domains as the skeleton of binding molecule assembling.The Fyn SH3 domains are can be in bacterium with high yield There is the complete human protein of 63 amino acid caused by rate.Fynomer can be connected together to obtain to two or more The different antigenic targets of kind have the multi-specific binding protein of compatibility.Fynomer is commercially available to be obtained from COVAGEN AG (Zurich, Switzerland).

It would be recognized by those skilled in the art that in the practice of method and composition claimed can by affine body or Fynomer is used as targeted molecular.

Bispecific and multi-specificity antibody

Bispecific antibody can be used in a variety of biomedical applications.For example, with thin to tumor cell surface antigen and T The bispecific antibody of the binding site of cellular surface acceptor can guide specific tumor cell to be dissolved by T cell.Identification nerve The bispecific antibody of CD3 epitopes in glioma and T cell has been successfully used to the brain tumor in treatment human patientses (Nitta etc., Lancet.1990；355：368-371).In certain embodiments, it is conjugated for therapeutic agent disclosed herein Technology and composition can be used as targeting moiety together with bispecific or multi-specificity antibody.

Numerous methods for producing bispecific or multi-specificity antibody are known, such as such as United States Patent (USP) No.7, Disclosed in 405,320, the embodiment part of the patent is incorporated herein by reference.Four body hybridoma sides can be passed through Method produces bispecific antibody, and methods described is related to the fusion of two kinds of different hybridomas, and the hybridoma each produces identification Monoclonal antibody (Milstein and the Cuello, Nature, 1983 of different antigen sites；305：537-540).

Chemical to tie two kinds of differences using heterobifunctional crosslinker for producing another method of bispecific antibody Monoclonal antibody (Staerz etc., Nature.1985；314：628-631；Perez etc., Nature.1985；316：354- 356).It can also then mix by two parental monoclonal antibodies are each reduced into corresponding half molecule and make its oxygen again Change and produce bispecific antibody (Staerz and Bevan.Proc Natl Acad Sci U S to obtain hybrid structure A.1986；83：1453-1457).Alternative dispensing means are directed to use with what appropriate joint independently purified two or three Fab ' fragments are chemically crosslinked.(see, for example, european patent application 0453082).

Other method is included by turning different selected markers via shuttle vector derived from retrovirus Corresponding parent hybridomas is moved to, is then merged (DeMonte etc., Proc Natl Acad Sci U S A.1990,87： 2941-2945)；Or hybridoma cell line is transfected with the expression plasmid of the heavy chain comprising different antibodies and light chain gene, to improve Produce the efficiency of hybrid hybridomas.

Can be with the peptide linker (being generally made up of more than 12 amino acid residues) with appropriate composition and length by homologous V_H And V_LDomain is bonded together to form the scFv (scFv) with binding activity.Manufacture scFv method is disclosed in the U.S. Patent No.4,946,778 and United States Patent (USP) No.5, in 132,405, its respective embodiment part is herein incorporated by reference this Wen Zhong.By peptide linker contraction in length to the V that can be prevented less than 12 amino acid residues on same chain_HAnd V_LDomain is matched and compeled Make the V on other chains with complementary domain_HAnd V_LDomain matches, so as to result in feature polymer.With with 3 to The V of the joint engagement of 12 amino acid residues_HAnd V_LThe polypeptide chain of domain primarily forms dimer (being referred to as double-chain antibody).Profit With the joint with 0 to 2 amino acid residue, advantageously form tripolymer (being referred to as three chain antibodies) and the tetramer (is referred to as four chains Antibody), but in addition to joint length, definite oligomerization pattern seems the composition and orientation (V depending on V structure domain_H- connect Head-V_LOr V_L- joint-V_H)。

For producing these technologies of polyspecific or bispecific antibody in low-yield, purifying necessity, low stability Or show all difficulties in terms of the labor-intensive of technology.Recently, using the technology for being referred to as " stop and locking (DNL) " come Produce it is substantially any needed for antibody, antibody fragment and other effector molecules combination (see, for example, United States Patent (USP) No.7,550, 143；7,521,056；7,534,866；7,527,787 and USSN11/925,408, its respective embodiment part is to quote Mode is incorporated herein).The technology is using being referred to as the mutual of anchoring domain (AD) and dimerization and dockerin domain (DDD) Benefit property protein binding domain, they are bonded to each other and realize the assembling of composite structure, and forming range includes dimer, trimerization Body, the tetramer, pentamer and six aggressiveness.These form stable compound without purifying extensively with high yield.DNL technologies permit Perhaps the assembling of monospecific, bispecific or multi-specificity antibody.It is used to manufacture bispecific or how special as is generally known in the art Property antibody any technology can be used in the practice of present invention method claimed.

Pre-targeting

Bispecific or multi-specificity antibody can be used in pre-targeting technology.Pre-targeting is to be originally developed to solve directly The multistage method that the slow blood of targeting antibodies is removed, its normal tissue such as marrow cause undesirable toxicity.By pre- Targeting, radionuclide or other therapeutic agents are connected to the small delivery molecule removed in several minutes from blood (can target Construct).Apply first pair can targeting construc and target antigen there is the pre-targeting bispecific or how special of binding site Property antibody so that free antibody is removed from circulation, then apply can targeting construc.

Pre-targeting method is disclosed in such as Goodwin et al., United States Patent (USP) No.4, and 863,713；Goodwin et al., J.Nucl.Med.29：226,1988；Hnatowich et al., J.Nuch Med.28：1294,1987；Oehr et al., J.Nucl.Med.29：728,1988；Klibanov et al., J.Nucl.Med.29：1951,1988；Sinitsyn et al., J.Nucl.Med.30：66,1989；Kalofonos et al., J.Nucl.Med.31：1791,1990；Schechter et al., Int.J.Cancer48：167,1991；Paganelli et al., Cancer Res.51：5960,1991；Paganelli et al., Nucl.Med.Commun.12：211,1991；United States Patent (USP) No.5,256,395；Stickney et al., Cancer Res.51： 6650,1991；Yuan et al., Cancer Res.51：3119,1991；United States Patent (USP) 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, each it is incorporated herein by reference.

The disease or the pre-targeting method of illness in treatment presented below or diagnosis subject can be passed through：(1) to subject Using bispecific antibody or antibody fragment；(2) optionally applied to the subject and remove composition, and make the combination Thing removes antibody from circulation；And (3) are applied comprising one or more chelating or chemically combined treatment to the subject Agent or diagnosticum such as interferon lambda can targeting construc.

Can targeting construc

In certain embodiments, may be selected by one or more therapeutic agents or diagnosticum mark for pre-targeting can Targeting construc peptide be incorporated into pair can targeting construc peptide have one or more binding sites and pair with disease or symptom phase The target antigen of pass has the bispecific antibody of one or more binding sites.Bispecific antibody can be used for pre-targeting technology In, wherein can be first to subject's administration of antibodies.Enough time can be allowed so that bispecific antibody be incorporated into target antigen and Uncombined antibody is set to be removed from circulation.Then can be applied to subject targeting construc such as labeled peptide and can make it It is incorporated into bispecific antibody and is positioned at diseased cells or tissue.

It is such can targeting construc can have and various structures and can target structure not only for being incorporated into high-affinity The antibody of body or the availability of fragment, and resist for working as in pre-targeting method and bispecific antibody (bsAb) or polyspecific Quick internal clearance rate is selected when using in vivo.Water-repelling agent, which is most good at, causes strong immune response, and hydrophilizing agent is excellent It is selected to rapid in vivo removing.Therefore, equilibrium establishment between hydrophobic characteristics and hydrophilic characteristics.This can be partially by use Hydrophily chelating agent offsets the intrinsic hydrophobicity of many organic moieties to realize.In addition, it may be selected that there is opposite dissolubility Matter can targeting construc subunit, such as peptide, it contains amino acid, some of them tool hydrophobicitys and some of tools are hydrophilic Property.

The peptide with as little as two amino acid residues, preferably 2 to 10 residues can be used, and it can also be coupled to other Part such as chelating agent.Joint should be low molecule amount conjugates, preferably has less than 50,000 dalton and is advantageously below about The molecular weight of 20,000 dalton, 10,000 dalton or 5,000 dalton.Can targeting construc peptide more generally will have four Or more residue, such as PEPD OTA-Phe-Lys (HSG)-Tyr-Lys (HSG)-NH₂(SEQ ID NO：98), wherein DOTA For Isosorbide-5-Nitrae, 7,10- tetraazacyclododecanand Isosorbide-5-Nitraes, 7,10- tetraacethyls and HSG is histamine succinyl base glycyl.Alternatively, DOTA can be by NOTA (Isosorbide-5-Nitrae, tri- azepines of 7--cyclononane-Isosorbide-5-Nitrae, 7- triacetic acids), TETA (to bromoacetamido-benzyl-triethylammonium tetrakis Tetraacethyl), NETA ([2- (4,7- double carboxymethyl group [Isosorbide-5-Nitrae, 7] 7-triazacyclononane -1- bases-ethyls] -2- carbonvlmethyls-ammonia Base] acetic acid) or the replacement of other known chelating moiety.Usable chelating moiety is for example to be incorporated into therapeutic and/or diagnostic put Penetrating property nucleic, paramagnetic ion or contrast agent.

Can targeting construc can also in backbone structure comprising alpha-non-natural amino acid such as D- amino acid to increase peptide in vivo Stability.In alternative embodiments, other backbone structures can be used, such as built by alpha-non-natural amino acid or class peptide Those backbone structures.

Solid phase support thing and the standard technique for repeating orthogonal deprotection and being coupled preferably is used to be closed on automated peptide synthesizer Into be used as can targeting construc peptide.The free amine group of conjugated chelating moiety or other agent will be used in peptide later advantageously Blocked with standard protecting group such as Boc groups, while the acetylation of N-terminal residue can be increased serum stability.Such protection group is It is well known to the skilled person.Referring to Greene and Wuts Protective Groups in Organic Synthesis, 1999 (John Wiley and Sons, N.Y).When peptide prepares to be later used in bispecific antibody system, They are advantageously cracked to generate corresponding C-terminal acid amides from resin, to suppress internal carboxypeptidase activity.The exemplary side of peptide symthesis Method is disclosed in Examples below.

When using bispecific antibody carry out pre-targeting when, antibody by comprising by target tissue produce or with target tissue association First binding site of antigen and the second binding site for haptens that can be in targeting construc.Exemplary haptens includes (but not limited to) HSG and In-DTPA.Recruitment is known (such as 679 antibody) to the antibody of HSG haptens and can be easily Be incorporated in appropriate bispecific antibody (see, for example, United States Patent (USP) No.6,962,702,7,138,103 and 7,300,644, close It is incorporated herein by reference in embodiment part).However, other haptens and to be incorporated into its antibody be in this area It is known and can be used, such as In-DTPA and 734 antibody (for example, United States Patent (USP) No.7,534,431, embodiment part It is incorporated herein by reference).

Therapeutic agent

In various embodiments, therapeutic agent for example cytotoxic agent, anti-angiogenic agent, promote apoptosis agent, antibiotic, swash Element, hormone antagonist, chemotactic factor (CF), medicine, prodrug, toxin, enzyme or other agent are used as interferon-antibody as described herein DNL^TMThe complementary therapy of construct.Medicine used, which can have, to be selected from by antimitotic agent, antikinase agent, alkylating agent, antimetabolic Thing, antibiotic, alkaloid, anti-angiogenic agent, promote apoptosis agent with and combinations thereof the pharmaceutical properties of group that form.

Illustrative drug used may include 5 FU 5 fluorouracil, APL (aplidin), azaribine (azaribine), Anastrozole (anastrozole), anthracycline, bendamustine (bendamustine), bleomycin (bleomycin), bortezomib (bortezomib), Bryostatin-1 (bryostain-1), busulfan (busulfan), thorn P0-357 (calicheamycin), camptothecine (camptothecin), carboplatin (carboplatin), 10-hydroxycamptothecine, card Mo Siting (carmustine), Celebrex (celebrex), Chlorambucil (chlorambucil), cis-platinum (cisplatin) (CDDP), Cox-2 inhibitor, Irinotecan (irinotecan) (CPT-11), SN-38, carboplatin, carat Qu Bin (cladribine), clofarabine (clofarabine), cytarabin (cytosine arabinoside), happiness Set bases, endoxan, cytarabine (cytarabine), Dacarbazine (dacarbazine), Docetaxel (docetaxel), dactinomycin D (dactinomycin), daunorubicin (daunorubicin), adriamycin (doxorubicin), 2- pyrrolinodoxorubicins (2P-DOX), cyano-morpholine base adriamycin, doxorubicin glucuronide, table are soft Than star glucosiduronic acid, Estramustine (estramustine), table podophyllotoxin (epipodophyllotoxin), ERs Bonding agent, Etoposide (etoposide) (VP16), Etoposide glucosiduronic acid, etoposide phosphate, floxuridine (FUdR), 3 ', 5 '-O- dioleoyls-FudR (FUdR-dO), fludarabine (fludarabine), Flutamide (flutamide), method Buddhist nun Base-protein transferase inhibitor, EGFR-TK and Bruton kinase inhibitors, gemcitabine (gemcitabine), hydroxyl Urea, idarubicin (idarubicin), ifosfamide (ifosfamide), leunase, lenalidomide (lenolidamide), formyl tetrahydrofolic acid (leucovorin), lomustine (lomustine), mustargen (mechlorethamine), melphalan (melphalan), purinethol, Ismipur, methotrexate (MTX) (methotrexate), mitoxantrone (mitoxantrone), mithramycin (mithramycin), mitomycin (mitomycin), mitotane (mitotane), NVB (navelbine), nitroso ureas, plicamycin (plicomycin), Procarbazine (procarbazine), taxol (paclitaxel), Pentostatin (pentostatin), PSI-341, Lei Luo Former times sweet smell (raloxifene), Semustine (semustine), streptozotocin (streptozocin), TAM (tamoxifen), taxol (taxol), Temozolomide (temazolomide) (DTIC aqueous form), anti-platinum (transplatinum), Thalidomide (thalidomide), thioguanine, thio-tepa (thiotepa), Teniposide (teniposide), TPT (topotecan), uracil mastard, vinorelbine (vinorelbine), vincaleukoblastinum (vinblastine), vincristine (vincristine) and vinca alkaloids.

Tyrosine kinase inhibitor used may include LFM-A13, Dasatinib (dasatinib), Imatinib Or AMN107 (nilotinib) (imatinib).

Toxin used may include ricin (WA), abrin, alpha toxin, saporin, ribalgilase (RNase) such as Ranpirnase, DNA enzymatic I, staphylococcus enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, pseudomonas Exotoxin and pseudomonas endotoxin.

Chemotactic factor (CF) used may include RANTES, MCAF, MIP1- α, MIP1- β and IP-10.

In certain embodiments, anti-angiogenic agent, such as angiostatin, bar chalone can be used (baculostatin), canstatin (canstatin), mammary gland silk suppression albumen (maspin), anti-VEGF antibody, anti-PlGF peptides With 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 peptide, fibronectin peptide, activator of plasminogen suppression Preparation, tissue inhibitor of metalloproteinase, interferon, interleukin 12, IP-10, Gro- β, thrombospondin, 2- Methoxyestradiol, proliferin GAP-associated protein GAP, carboxyl acyl aminotriazole (carboxiamidotriazole), CM101, horse are pulled up a horse Take charge of his (Marimastat), pentosane polysulfate ester, angiopoietin-2, interferon, Antibiotic TAN 420F, PNU145156E, 16K Prolactin(PRL fragment, linomide (roquinimex), Thalidomide, PTX, genistein, TNP-470, endostatin, Taxol, accutin, angiostatin, cidofovir (cidofovir), vincristine, bleomycin, AGM-1470, blood are small The plate factor 4 or minocycline (minocycline).

Other useful therapeutic agents can include oligonucleotides, particularly preferably be directed to oncogene and oncogene products Such as bcl-2 or p53 ASON.A kind of preferable therapeutic oligonucleotide form is siRNA.

Diagnosticum

Diagnosticum is preferably chosen from by radionuclide, radiocontrast medium, paramagnetic ion, metal, fluorescence labeling, chemistry The group of luminescent marking, acoustic contrast agent and sensitising agent composition.The diagnosticum be it is well known that and can be used it is any this The known diagnosis agent of sample.The non-limiting examples of diagnosticum may include radionuclide, such as¹¹⁰In、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 γ hairs Beam, beta emitter or positron emitter.Paramagnetic ion used may include chromium (III), manganese (II), iron (III), iron (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 may include lanthanum (III), golden (III), lead (II) or bismuth (III).Acoustic contrast agent can Liposome is such as inflated comprising liposome.Radiopaque diagnosticum may be selected from compound, barium compound, gallium compound and thallation and close Thing.Extensive a variety of fluorescence labelings, including but not limited to fluorescein isothiocynate, rhodamine, phycoerythrin as is generally known in the art (phycoerytherin), phycocyanin, allophycocyanin, OPA and fluorescamine.Available chemiluminescent labeling can Including luminol, different luminol, aromatics acridinium ester, imidazoles, acridinium salt or oxalate.

Conjugated technology

In certain embodiments, can be by DNL^TMConstruct is conjugated in one or more therapeutic agents or diagnosticum.For example, can Will¹³¹I is incorporated to the tyrosine of protein or peptide, or is connected to the medicine of the ε amino of lysine residue.By therapeutic agent and it can also examine Disconnected agent is connected to the SH groups of such as reduction.It is a variety of as is generally known in the art to prepare being total to for therapeutic agent or diagnosticum and protein or peptide The method of valency or non-covalent conjugates, and using any such known method.

Heterobifunctional crosslinker such as 3- (2- pyridyidithios) propionic acid N- succinyl base esters (SPDP) connection can be used to control Treat agent or diagnosticum.Yu etc., Int.J.Cancer56：244(1994).It is well known in the art for such a conjugated general Technology.See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press1991)；Upeslacis etc., " Modification of Antibodies by Chemical Methods, " MONOCLONAL ANTIBODIES：The (eds.) such as PRINCIPLES AND APPLICATIONS, Birch, the 187-230 pages (Wiley-Liss, Inc.1995)；Price, " Production and Characterization of Synthetic Peptide-Derived Antibodies, " MONOCLONAL ANTIBODIES：PRODUCTION, ENGINEERING AND The (eds.) such as CLINICAL APPLICATION, Ritter, the 60-84 pages (Cambridge University Press1995).

In some embodiments, chelating agent can be connected to protein or peptide and be used for chelating therapy agent or diagnosticum, Such as radionuclide.Exemplary chelators include but is not limited to DTPA (such as Mx-DTPA), DOTA, TETA, NETA or NOTA.Method well known in the art conjugated and that metal or other parts and protein or peptide are connected using chelating agent (see, for example, United States Patent (USP) No.7,563,433, embodiment part is incorporated herein by reference).It is particularly useful Metal-chelate combination include 2- benzyl-DTPA and its monomethyl and cyclohexyl analogs, can with general energy range for 60 to 4,000keV diagnosis isotope is used for radiological imaging together, and the isotope is for example¹²⁵I、¹³¹I、¹²³I、¹²⁴I、⁶²Cu、⁶⁴Cu、¹⁸F、¹¹¹In、⁶⁷Ga、⁶⁸Ga、^99mTc、^94mTc、¹¹C、¹³N、¹⁵O or⁷⁶Br.Answered when with non-radioactive metal such as magnesium, iron and gadolinium During conjunction, identical chelate can be used for MRI.Macrocyclic chelate thing such as NOTA, DOTA and TETA can be with various metals and radiogold Category is used together, and more particularly the radionuclide respectively with gallium, iridium and copper is used together.Can be by for interested metal The size of customization ring makes the metal-chelate complexes highly stable.Cover other ring-like chelate such as macrocyclic polyethers, its Available for stable bond nucleic, such as in RAIT²²³Ra。

Recently, it has been disclosed that used in PET scan technology¹⁸The method of F marks, for example, by by F-18 and metal or other Atom such as aluminium is reacted.¹⁸F-Al conjugates can target with being connected directly to antibody or for being marked in pre-targeting method The chelation group of construct such as DOTA, NOTA or NETA is compound.The F-18 labelling techniques are disclosed in United States Patent (USP) No.7,563, In 433.

Treatment method

Various embodiments be related to subject such as mammal (including the mankind, domestic or companion pet, such as dog and Cat) in treating cancer method, it include to the subject apply therapeutically effective amount interferon-antibody DNL^TMConstruct. Can be right by simultaneously or sequentially applying the antigen being incorporated on target cell surface of therapeutically effective amount or the antibody reacted Interferon-antibody DNL^TMThe administration of construct is supplemented.Other preferable MAb include it is at least one be selected from by with following reaction MAb compositions group humanization, 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 be by simultaneously or sequentially further supplementing interferon-antibody DNL using at least one therapeutic agent^TMBuild physical exercise therapy Method.Such as " CVB " (1.5g/m²Endoxan, 200-400mg/m²Etoposide and 150-200mg/m²BCNU) be for Treat the scheme of NHL.Patti et al., Eur.J.Haematol.51：18(1993).Those skilled in the art It is known that other suitable joint chemotherapeutic treatment protocols.See, for example, Freedman etc., " Non-Hodgkin ' s Lymphomas, " CANCER MEDICINE, volume 2, the 3rd edition, the (eds.) such as Holland, the 2028-2068 pages (Lea＆ Febiger1993).As explanation, for treating the first generation chemotherapeutic treatment protocols bag of intergrade NHL (NHL) Include C-MOPP (endoxan, vincristine, procarbazine and prednisone) and CHOP (endoxan, adriamycin, vincristine and Prednisone).Useful second generation chemotherapeutic treatment protocols be m-BACOD (methotrexate (MTX), bleomycin, doxorubicin, cyclophosphamide, Vincristine, dexamethasone and formyl tetrahydrofolic acid), and suitable third generation scheme be MACOP-B (methotrexate (MTX), adriamycin, Endoxan, vincristine, prednisone, bleomycin and formyl tetrahydrofolic acid).Other useful medicines include phenyl butyrate, Bendamustine (bendamustine) and Bryostatin-1.

Interferon-antibody DNL can be prepared according to known method^TMConstruct to prepare the composition of pharmaceutically useful, according to This is by interferon-antibody DNL^TMConstruct is with pharmaceutically suitable excipient composition in mixture.Sterile phosphate buffer salt Water is an example of pharmaceutically suitable excipient.Those skilled in the art are it is known that other suitable excipient.Referring to Such as Ansel etc., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, the 5th edition (Lea＆ Febiger1990), and Gennaro (eds.), REMINGTON ' S PHARMACEUTICAL SCIENCES, the 18th edition (Mack Publishing Company1990), and its revised edition.

Can be by interferon-antibody DNL^TMConstruct is formulated for intravenously applying, for example, by bolus infusion or continuous defeated Liquid.Preferably, by interferon-antibody DNL^TMPeriod of the construct through less than about 4 hours and more preferably through less than about 3 The period transfusion of hour.For example, initial 25-50mg can infuse in 30 minutes, preferably even 15 minutes, and remaining part Infused through subsequent 2-3 hours.Ejection preparation can be presented with unit dosage forms, for example, in ampoule or multi-dose container, wherein adding There is preservative.Composition can be used in suspension, solution or emulsion form in oiliness or aqueous vehicles, and can include Preparaton such as suspending agent, stabilizer and/or dispersant.Alternatively, active component can be powder type, be carried before use with suitable For example sterile apirogen water reconstruct of body.

Other pharmaceutical methods can be used to control interferon-antibody DNL^TMThe duration of construct effect.Can be by using Polymer is compound or adsorptive hindrance it is plain-antibody DNL^TMConstruct prepares control release preparation.For example, biocompatible polymer Polyanhydride copolymer matrix including poly- (ethylene-co-vinylacetate) matrix and stearic acid dimer and decanedioic acid. Sherwood etc., Bio/Technology10：1446(1992).The rate of release of the matrix depends on interferon-antibody DNL^TMMolecular weight, the Medium Culture interferon-antibody DNL of construct^TMThe amount of construct and the size of dispersed particle. Saltzman etc., Biophys.J.55：163(1989)；Sherwood etc., ibid.Other solid dosage forms are described in Ansel etc., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, the 5th edition (Lea＆Febiger1990), With Gennaro (eds.), REMINGTON ' S PHARMACEUTICAL SCIENCES, the 18th edition (Mack Publishing Company1990) and its described in revised edition.

Interferon-antibody DNL^TMConstruct can also carry out subcutaneous administration or even other parenteral routes to mammal Using.Period of the construct preferably through less than about 4 hours and the period transfusion more preferably through less than about 3 hours.

In the case of more common, interferon-antibody DNL of the mankind is applied to^TMThe dosage of construct will be according to such as patient The factors such as age, body weight, height, sex, general medicine symptom and prior medical history and change.In non-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.If it would be recognized by those skilled in the art that see Toxicity sign is observed, then can reduce dosage or stopped treatment.If desired, repeatable administration, for example, continuing 4-10 twice a week It is all, weekly to continue 4-10 weeks, continue 8 weeks once in a week or continue 4 weeks once in a week.Can also be according to the need of maintenance therapy To be administered with smaller frequency, such as continue some months once every two weeks, or every month or each season once continue multiple moons. It is alternatively possible to every 2 weeks or 3 weeks doses apply interferon-antibody DNL^TMConstruct, repeat at least 3 dosage altogether.Or Person, construct can be administered twice weekly and continue 4-6 weeks.According to the appropriate regulation to dosage and scheme, dosage regimen is optionally Repeated with other intervals, and various parenteral administrations can be passed through.

In preferred embodiments, interferon-antibody DNL^TMConstruct can be used for treatment of cancer.The example of cancer includes (but not limited to) carcinoma, lymthoma, glioblastoma, melanoma, sarcoma and leukaemia, myeloma or lymphatic system are disliked Property tumour.The more specific examples of the cancer indicate in it is lower and including：It is squamous cell carcinoma (for example, epithelial squamous cell cancer), outstanding Because of sarcoma, the nephroblastoma, astrocytoma, lung cancer (including ED-SCLC, non-small cell lung cancer, adenocarcinoma of lung and lung squama Cancer), peritoneal cancer, hepatocellular carcinoma, stomach cancer (gastric cancer) or stomach cancer (stomach cancer) (including human primary gastrointestinal cancers), pancreas Gland cancer, glioblastoma multiforme, cervix cancer, oophoroma, liver cancer, carcinoma of urinary bladder, hepatoma, hepatocellular carcinoma, nerve in Secreting tumor, medullary carcinoma of thyroid gland, differentiated thyroid carcinoma, breast cancer, oophoroma, colon and rectum carcinoma, carcinoma of endometrium or Uterine cancer, salivary-gland carcinoma, kidney (kidney cancer) or kidney (renal cancer), prostate cancer, carcinoma of vulva, anus Cancer, carcinoma of penis and head and neck cancer.Term " cancer " includes primary malignancy cell or tumour (for example, cell does not move to also The malignant tumour or tumour of the interior other positions in addition to initial malignant tumour or knub position of subject's body) and Secondary cases Malignant cell or tumour are (for example, from metastatic tumor, malignant cell or tumor cell migration to different from initial knub position Secondary cases position malignant cell or tumour).

Other of cancer or malignant tumour example include but is not limited to：Children acute lymphoblastic leukemia, urgency Property lymphoblastic leukemia, acute lymphatic leukemia, acute myelocytic leukemia, adrenocortical carcinoma, into People (primary) hepatocellular carcinoma, adult (primary) liver cancer, adult acute's lymphocytic leukemia, adult acute's myeloid Leukaemia, adult's Hodgkin lymphoma, adult lymphoid cell leukemia, adult's NHL, Adult Primary liver Cancer, adult soft tissue sarcoma, AIDS associated lymphomas, AIDS associated malignancies, cancer of anus, astrocytoma, cholangiocarcinoma, wing Guang cancer, osteocarcinoma, brain stem glioma, brain tumor, breast cancer, renal plevis and carcinoma of ureter, central nervous system (primary) leaching Bar knurl, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma, cervix cancer, children (primary) liver are thin Born of the same parents' cancer, children (primary) liver cancer, children acute lymphoblastic leukemia, children acute myelocytic leukemia, children Brain stem glioma, Cerebellar Astrocytoma in Children. An, children's cerebral astrocytoma, children's extracranial germ cell knurl, children are suddenly Strange gold disease, study on Hodgkin lymphoma in children, children's hypothalamus and pathways for vision glioma, the white blood of children lymphoblast property Disease, Children Medulloblastoma, Non-Hodgkin Lymphoma in Children, children's pineal body and Supratentorial primitive neuroectodermal tumour, children It is primary carcinoma of liver, Children Rhabdomyosarcoma, children soft tissue sarcoma, children's vision path and inferior colliculus glioma brain tumour, chronic Lymphocytic leukemia, chronic granulocytic leukemia, colon cancer, skin T cell lymphoma, endocrine islet cell cancer, Carcinoma of endometrium, ependymoma, epithelioma, cancer of the esophagus, Ewing's sarcoma and related neoplasms, exocrine pancreas cancer, the outer reproduction of cranium Cytoma, Extaagonactal perm celi tumors, cholangiocarcinoma, cancer eye, women with breast cancer, gaucher's disease (Gaucher ' s Disease), gallbladder cancer, stomach cancer, stomach and intestine carcinoid tumor, stomach and intestine tumor, germinoma, gestational trophoblastic neoplasms, hair Chronic myeloid leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin lymphoma, hypergammaglobulinemia, hypopharyngeal cancer, intestinal cancer, intraocular melanin Knurl, islet-cell carcinoma, islet cells cancer of pancreas, Kaposi sarcoma, kidney, laryngocarcinoma, lip and carcinoma of mouth, liver cancer, lung cancer, lymph Proliferative disorders, macroglobulinemia, male breast carcinoma, malignant mesothelioma, malignant thymoma, medulloblastoma, melanoma, Celiothelioma, the invisible primary squamous cell neck cancer of transfer, metastatic primary squamous cell neck cancer, metastatic squamous cell neck Cancer, Huppert's disease, Huppert's disease/thick liquid cell anything superfluous or useless, myelodysplastic syndrome, myelocytic leukemia, bone Myelogenous leukemia, myeloproliferative illness, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, NHL, Nonmelanoma skin cancer, non-small cell lung cancer, invisible primary metastatic squamous cell neck cancer, oropharyngeal cancer, bone/pernicious fibre Tie up sarcoma, osteosarcoma/MFH, osteosarcoma/malignant fibrous histiocytoma of bone, epithelial ovarian cancer, ovary Germinoma, low grade of malignancy ovarian neoplasm, cancer of pancreas, paraproteinemia, polycythemia vera, parathyroid gland Cancer, carcinoma of penis, pheochromocytoma, hypophysoma, primary central nervous system lymphoma, primary carcinoma of liver, prostate cancer, rectum Cancer, clear-cell carcinoma, renal plevis and carcinoma of ureter, retinoblastoma, rhabdomyosarcoma, salivary-gland carcinoma, sarcoidosis sarcoma, Sai Zha In syndrome, cutaneum carcinoma, ED-SCLC, carcinoma of small intestine, soft tissue sarcoma, squamous cell neck cancer, stomach cancer, original nerve is outer on curtain Germinal layer and pinealoma, t cell lymphoma, carcinoma of testis, thymoma, thyroid cancer, transitional cell carcinoma, the kidney of renal plevis and ureter Broad-mouthed receptacle for holding liquid and ureter are divided a word with a hyphen at the end of a line cancer, trophoblastic tumor, ureter and renal plevis cell cancer, carcinoma of urethra, uterine cancer, sarcoma of uterus, vagina Cancer, pathways for vision and inferior colliculus glioma brain tumour, carcinoma of vulva, Waldenstrom's macroglobulinemia (Waldenstrom ' s Macroglobulinemia), the nephroblastoma, and any other in tract listed above in addition to anything superfluous or useless Excessively proliferative disease.

Method and composition described herein and claimed can be used for treating pernicious or premalignant symptom and preventing Progress turns into anything superfluous or useless or malignant tumor state, including but not limited to those described above illness.The purposes is known or doubtful Seemingly continue to progress to have been indicated in the symptom of anything superfluous or useless or cancer, particularly include hyperplasia, change occurs in non-anything superfluous or useless cell growth (summary of the misgrowth situation is referring to Robbins and Angell, Basic when life or most particularly depauperation Pathology, second edition, 68-79 pages of W.B.Saunders Co., Philadelphia, the (1976)).

Depauperation is often the omen of cancer, and is mainly found in epithelium.It is non-neoplastic cell growth in most without A kind of form of sequence, including lose the uniformity of single cell and lose the structure orientation of cell.Depauperation is typically sent out Life is at the position that chronic stimulation or inflammation be present.Medicable depauperation illness includes but is not limited to anhidrotic ectodermal hair Educate bad, front face depauperation (anterofacial dysplasia), asphyxiating thoracic dysplasia, atrium-refer to development Bad, broncho-pulmonary dysplasia, cerebral dysplasia, cervical dysplasias are bad, chondroectodermal dysplasia, clavicle calvarial bone Bad, congenital ectodermal dysplasia, skull backbone dysplasia (craniodiaphysial dysplasia), cranium wrist plantar Osteodysplasty, skull metaphysis hypoplasia (craniometaphysial dysplasia), dentine depauperation, backbone It is depauperation, ectodermal dysplasia, enamel depauperation, encephalo-ophthalmic dysplasia, dysplasia epiphysialis hemimelia, multiple Property epiphyseal dysplasia, chondrodystrophia congenita punctata, epithelial dysplasia, face-refer to (toe)-genital development are bad, familial Jawbone fibrous dysplasia, familial white pleat sexual organ dysplasia, Fibromuscular dysplasia, bone fibres sexual development are not The outer embryo of good, florid osseous dysplasia, heredity kidney retinal dysplasia, hidrotic ectodermal dysplasia, hypohidrosis Layer depauperation, lymphocyte reduction property thymic hypoplasia, mammary dysplasia, lower maxillofacial bone depauperation, metaphysis Depauperation, Mondini depauperation, monostotic fibrous dysplasia, mucous epithelium depauperation, the development of multiple epiphysis Bad, ocular spine dysplasia, oculodentodigital dysplasia, eye vertebral are bad, tooth source sexual organ dysplasia, now jaw development Bad, periapex cemental dysplasia, polyostotic fibrodysplasia, the development of pseudoachondroplasia vertebra epiphysis are not Good, retinal dysplasia, Septo-optic dysplasia, spondyloepiphyseal dysplasia and ventricles of the brain radial direction depauperation.

Other medicable preneoplasia illnesss include but is not limited to hyperplasia of prostate disorder (for example, benign tumour, Fibrocyst symptom, tissue hyperplasia, intestinal polyp or adenoma and oesophagus depauperation), leukoplakia, seborrheic keratosis, bowen's disease (Bowen ' s disease), farmer's skin disease (Farmer ' s Skin), solar cheilitis and solar keratosis.

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

Other excessively proliferative diseases, illness and/or symptom include but is not limited to malignant tumour and the progress of associated conditions And/or transfer, such as leukaemia (including acute leukemia is (for example, acute lymphatic leukemia, acute myelocytic are white Blood disease (including myeloblastic, promyelocytic leukemic cell, myelo-monocytic, monocarpotic cellularity and erythroleukemia)) and it is (chronic white Blood disease (for example, chronic myeloid (granulocytic) leukaemia and chronic lymphocytic leukemia))), genuine erythrocyte increase More diseases, lymthoma (for example, Hodgkin's disease and non-Hodgkin lymphoma), Huppert's disease, Waldenstrom's macroglobulinemia, heavy chain disease, And entity tumor, including but not limited to sarcoma and carcinoma such as fibrosarcoma, myxosarcoma, embryonal-cell lipoma, chondrosarcoma, bone and flesh Knurl, chordoma, angiosarcoma, endotheliosarcoma, lymphangioendothelial sarcoma, lymphangioendothelial sarcoma, synovialoma, celiothelioma, Ewing' s tumor, Leiomyosarcoma, rhabdomyosarcoma, colon cancer, cancer of pancreas, breast cancer, oophoroma, prostate cancer, squamous cell carcinoma, substrate are thin Born of the same parents' cancer, gland cancer, syringocarcinoma, carcinoma of sebaceous glands, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, cephaloma, bronchiolar carcinoma, nephrocyte Cancer, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, the nephroblastoma, cervix cancer, orchioncus, lung Cancer, ED-SCLC, carcinoma of urinary bladder, epithelioma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, endyma Knurl, pinealoma, hemangioblastoma (emangioblastoma), acoustic neurinoma, few prominent glioma, meningioma, black Plain knurl, neuroblastoma and retinoblastoma.

In other embodiments, Pegylation DNL^TMCompound can be used for treatment pathogenic infection organism such as bacterium, Virus or the subject of fungi.Medicable exemplary fungi includes Microsporon (Microspomm), trichophyton (Trichophyton), Epidermophyton (Epidermophyton), Sporothrix schenckii (Sporothrix schenckii), Cryptococcus neoformans (Cryptococcus neoformans), posadasis spheriforme (Coccidioides immitis), capsule tissue Endochylema bacterium (Histoplasma capsulatum), Blastomyces dermatitidis (Blastomyces dermatitidis) or Candida albicans Bacterium.Exemplary viral include human immunodeficiency virus (HIV), herpesviral, cytomegalovirus, rabies viruses, influenza virus, HPV, hepatitis type B virus, HCV, sendai virus, feline leukaemia virus, reovirus, The tiny sample virus of poliovirus, human serum, simian virus 40, Respiratory Syncytial Virus(RSV), mouse mammary tumor virus, Varicella virus, dengue virus, rubella virus, measles virus, adenovirus, human T-leukemia virus, love are sprinkled Si Tan-epstein-Barr virus, Murine Leukemia Virus, mumps virus, vesicular stomatitis virus, sindbis alphavirus, lymphocyte Property choriomeningitis virus or blue tongue rims.Exemplary bacterium include Bacillus anthracis (Bacillus anthracis), Streptococcusagalactiae, legionella pneumophilia, micrococcus scarlatinae, Escherichia coli, NEISSERIA GONORRHOEAE, Neisseria meningitidis, pneumococcus Category, H influenzae type B, treponemal bacterium, lyme disease spirochete, pseudomonas aeruginosa, Mycobacterium leprae, miscarriage cloth Shandong Salmonella, mycobacterium tuberculosis or mycoplasma.

Kit

Various embodiments can relate to comprising the kit for being suitable for the part for treating patient's tissue.It is exemplary Kit can contain at least one or more of interferon-antibody construct as described herein.If contain and apply part Composition be not configured to deliver by alimentary canal, such as oral delivery, then may include in kit can by some its The device of his approach delivery of agents box part.A type of for applying non-bowel delivery apparatus is such as by composition The syringe that injection enters in subject's body.Suction apparatus can also be used.In certain embodiments, sterile liquid can be contained The form of the precharging type syringe or automatic injection pen of preparation or lyophilized formulations provides therapeutic agent.

Each part of kit can be packaged together or be divided in two or more containers.In some embodiment party In case, container can be the phial of the sterile freeze-drying preparation containing the composition suitable for reconstruct.Kit can also include it is a kind of or A variety of buffer solutions for being suitable to reconstruct and/or diluting other reagents.Other available containers include but is not limited to capsule, disk, box, Pipe etc..Kit forms part in container inner packing and can keep sterile.Another part that may include makes for people With the specification of kit.

Embodiment

The claim that following examples are used to illustrating but not limiting the present invention is provided.

Embodiment 1.C_H3- AD2-IgG expression vectors

PdHL2 mammalian expression vectors have been used for expression restructuring IgG (Qu etc., Methods2005,36：84-95). A kind of plasmid shuttle vector is produced to aid in any IgG-pdHL2 carriers being changed into C_H3- AD2-IgG-pdHL2 carriers.Use PdHL2 carriers expand Fc (C as template and using following Oligonucleolide primers by PCR_H2And C_H3Domain) gene：

Fc BglII are left

AGATCTGGCGCACCTGAACTCCTG(SEQ ID NO：90)

Fc Bam-EcoRI are right

GAATTCGGATCCTTTACCCGGAGACAGGGAGAG(SEQ ID NO：91).

Amplimer is cloned in pGemT PCR cloning vectors (Promega).Fc is inserted using Xba I and Bam HI Enter fragment by pGemT shear and with by using Xba I and Bam HI digest h679-Fab-AD2-pdHL2 (Rossi etc., Proc Natl Acad Sci USA2006,103：6841-6) the AD2-pdHL2 carriers prepared are connected to generate shuttle vector Fc-AD2-pdHL2.In order to which IgG-pdHL2 expression vectors are converted into C_H3- AD2-IgG-pdHL2 expression vectors, by 861bp's BsrG I/Nde I restricted fragments shear off from the former and use shearing from the 952bp of Fc-AD2-pdHL2 carriers BsrG I/Nde I restricted fragments are replaced.C to have generated and for producing recombinant humanized IgG-AD2 modules below_H3-AD2-IgG- The partial list of pdHL2 expression vectors：

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_H3-AD2-IgG generation

Stable C_HThe transfection and selection of the thin bag system of 3-AD2-IgG secretions

All cell line grows in hybridoma SFM (Invitrogen, Carlsbad CA).By using Sal I Digestion with restriction enzyme simultaneously enters Sp2/0-Ag14 (2.8 × 10 by (450 volts, 25 μ F) transfections of electroporation⁶Individual cell) will C_H3- AD2-IgG-pdHL2 carriers (30 μ g) linearize.The pdHL2 carriers include the gene of dihyrofolate reductase, it is allowed to make Immune Clone Selection and gene magnification are carried out with methotrexate (MTX) (MTX).

After transfection, by cell on 96 orifice plates bed board and in the culture medium containing 0.2 μM of MTX select transgenosis gram It is grand.The C of each clone is screened by using the sandwich ELISA of 96 hole microtiter plates of specific anti-idiotypic MAb coatings_H3- AD2-IgG productivity ratio.The Conditioned immunolresponse for estimating clone is transferred in microplate wells, and uses horseradish peroxidase yoke The Goat anti human class IgG F (ab ') of conjunction₂(Jackson ImmunoResearch Laboratories, West Grove, PA) Detection fusion albumen.The hole amplification of highest signal will be provided and eventually for production.

C_HThe generation and purifying of 3-AD2-IgG modules

In order to produce fusion protein, with 2 × 10⁵Individual cell/ml density sows cell in roller bottle culture and at 37 DEG C And 5%CO₂Under in roller bottle incubator incubate until cell survival rate fall below 25% (about 10 days).Will culture by centrifugation Meat soup is clarified, and is filtered and is concentrated by ultrafiltration to 50 times.In order to purify C_H3-AD2-IgG modules, by the supernatant loading of concentration Onto albumin A (MAB Select) affinity column.The post is washed till baseline using PBS and uses 0.1M glycine (pH2.5) will The fusion protein elutes.

The generation for the Fab and IgG fusion proteins that AD of the embodiment 3. from Multiple Antibodies connects with DDD

Using the technology described in previous embodiment, IgG and Fab fusion proteins shown in structure table 6 and DNL is incorporated to In construct.The fusion protein remains the antigenic binding property of parental antibody and DNL constructs show be incorporated to it is anti- The antigen-binding activity of body or antibody fragment.

Table 6. includes IgG or Fab fusion protein

Embodiment 4. is based on the generation of interferon (IFN)-α 2b DDD modules

For the IFN-α 2b-DDD2-pdHL2 expressed in mammalian cell structure

IFN-α 2b cDNA sequence is expanded by PCR, obtains including the sequence of following characteristics, wherein XbaI and BamHI are Restriction site, signal peptide is IFN-α 2b natural signals peptides, and 6His is hexahistidine tag：XbaI--- signal peptides --- (6His is with SEQ ID NO by IFN α 2b---6His---BamHI：92 disclosures).Gained secretory protein in its C-terminal by merging following sequence The IFN-α 2b compositions of the polypeptide of row：

KSHHHHHHGSGGGGSGGGCGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO：93).

Use total length mankind IFN α 2b cDNA clones (the human cloned catalogue # of InVitrogen Ultimate ORF HORF01 clones ID IOH35221) be template and following oligonucleotide is that primer realizes PCR amplifications：

IFNA2Xba I are left

TCTAGACACAGGACCTCATCATGGCCTTGACCTTTGCTITACTGG(SEQ ID NO：94)

IFNA2BamHI is right

GGATCCATGATGGTGATGATGGTGTGACTTTTCCTTACTTCTTAAACTTTCTTGC(SEQ ID NO：95)

Pcr amplification primer thing is cloned in pGemT carriers.The following DDD2-pdHL2 prepared for connecting IFN-α 2b is fed Newborn animal expression vector.Using 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, remove all Fab gene orders but retain DDD2 coded sequences. IFN-α 2b amplimers are sheared from pGemT and are connected on DDD2-pdHL2 carriers to generate using Xba I and Bam HI Expression vector IFN-α 2b-DDD2-pdHL2.

IFN-α 2b-DDD2 mammalian cell expression

IFN-α 2b-DDD2-pdHL2 is digested using Sal I and linearized, and Sp/ is stably transfected into by electroporation (referring to U.S. Patent application 11/877,728, embodiment part is hereby incorporated herein by ESF myeloma cell In).Two IFN-α 2bs of the clone with detectable level are found using ELISA.One of the two clones, are named as 95, can be Adapt to grow in serum free medium in the case of not significantly reducing productivity ratio.After through five weeks by the clone with 0.1 to 0.8 The MTX amplifications of μM progressive concentration.In this stage, it is subcloned by limiting to dilute and is expanded the subclone (95-5) of most high yield Increase.The use of business rIFN- α 2b (Chemicon IF007, lot number 06008039084) is reference material, the 95- being grown in shaking flask 5 estimated output is 2.5mg/L.

IFN-α 2b-DDD2 is purified in the batch culture grown from roller bottle

Clone's 95-5 amplifications are contained in 20L Serum-free Hybridomas SFM and 0.8 μM of MTX roller bottle altogether and made to 34 Arrival latter stage culture.By culture broth processing and as follows by immobilization metal affinity chromatography (IMAC) by IFN-α 2b- DDD2 is purified.Supernatant is clarified by centrifugation, 1X combination buffers (10mM imidazoles, 0.5M are arrived in 0.2 μM of filtering, diafiltration NaCl、50mM NaH₂PO₄, pH7.5) in, 310mL is concentrated into, Tween20 is added and reaches ultimate density 0.1%, and be loaded to On 30mL Ni-NTA posts.After sample loading, successively with 0.02%Tween20s of the 500mL in the 1X combination buffers and 290mL 30mM imidazoles, 0.02%Tween20,0.5M NaCl, 50mM NaH₂PO₄(pH7.5) column scrubber.Use 110mL's 250mM imidazoles, 0.02%Tween20,150mM NaCl, 50mM NaH₂PO₄(pH7.5) by product elution.Purifying obtains about 6mg IFNα2b-DDD2。

Generations of the IFN-α 2b-DDD2 in Escherichia coli

Also can by IFN-α 2b-DDD2 in the form of soluble protein by microbial fermentation in expression in escherichia coli.Use IFN-α 2b-DDD2-pdHL2 DNA are that template expands coded sequence by PCR.Use Nde I and Xho I restriction sites The amplimer is cloned into pET26b coli expression carriers.By being entered at 18 DEG C using 100 μm of IPTG to LB shaking flasks 12 hour inductions of row express protein into the cell in BL21pLysS host cells.Can as described above by IMAC Dissolubility IFN-α 2b-DDD2 purifies from cell lysates.

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

Following preparation, which includes, is connected to C_H3The DNL compounds (Fig. 1) of-AD2-IgG four IFN-α 2b-DDD2 parts.Letter For wanting, by selected C_H3-AD2-IgG combines with the IFN-α 2b-DDD2 of about two molar equivalents, and add 1mM EDTA and After 2mM reduced glutathiones (GSH), the mixture is reduced in ambient temperature overnight under mild conditions.Add oxidized form paddy The sweet peptide of Guang is placed in room temperature 12-24 hours again to 2mM and by the mixture.The DNL conjugates is purified by albumin A affinity column.System Standby four kinds of such DNL conjugates for being designed to 20-2b, 22-2b, hR1-2b and 243-2b, respectively include the difference of four copies It is anchored to C_H3-AD2-IgG-hA20 (there is CD20 specificity), C_H3-AD2-IgG-hLL2 (there is CD22 specificity), CH3- AD2-IgG-hR1 (there is IGF-1R specificity) and C_HIFN-α on 3-AD2-IgG-hL243 (there is HLA-DR specificity) 2b.The 20-2b analytical tables generated using SE-HPLC to IFN-α 2b-DDD2 caused by mammal (m) or Escherichia coli (e) Bright, an every kind of main peak for showing that the holdup time is consistent with the covalent complex being made up of IgG and 4 IFN-α 2b group is (not Show).Similar SE-HPLC overviews are observed in other three kinds of IFN-IgG conjugates.

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

20-2b external IFN α is given birth to using the reporter based on cell, virus protection and lymthoma proliferation assay Thing activity is compared with 2 doses of PEGASYS and PEG- intrones of business Pegylation IFN α.Use the kit based on cell Activity specific is determined, it utilizes monokaryon before the transgenosis mankind with the reporter for being blended in IFN-stimulated responsive element Cell line (Fig. 2A -2D).20-2b (5300IU/pmol) activity specific is more than in PEGASYS (170IU/pmol) and PEG- Containing sub (3400IU/pmol) (Fig. 2A).With 734-2b, 1R-2b caused by the method similar to 20-2b and five kinds of other MAb- IFN α construct (data are not shown), similar activity specific (4000-8000IU/pmol) is shown, shows to generate this The uniformity (Fig. 2A) of the DNL methods of class formation.Contribute to strengthen MAb-IFN α effect with four IFN α 2b groups.When right After the normalization of IFN α equivalent, activity specific/IFN α is higher than PEGASYS about 10 times, and only lower than PEG- introne about 2 Times.

MAb-IFN α, PEGASYS and PEG- intrones are compared in virus protection determination method in vitro, show MAb- IFN α retains the IFN α 2b antiviral activities (Fig. 2 B) of the activity specific similar to PEG- intrones and for PEGASYS10 times.

IFN α 2b can have direct antiproliferative or cytotoxic effect to some tumor cell lines.Proliferation assay in vitro With the measurement (Fig. 2 C) to the extremely sensitive Burkitt lymphoma cell line of IFN α (Daudi) progress 20-2b activity in method.Each 2 doses of IFN α is in vitro with efficient (EC₅₀=4-10pM) effectively suppress (＞ 90%) Daudi.However, 20-2b (EC₅₀= It is 0.25pM) more more potent about 30 times than non-targeted MAb-IFN α constructs.(the EC under at a relatively high concentration₅₀＞ 10nM), 20- The 2b anti-CD20MAb of parent in a variety of lymphoma cell lines (including Daudi) have external antiproliferative activity (Rossi etc., 2008, Cancer Res68：8384-92).Also 20-2b external activity is assessed using Jeko-1, Jeko-1 is to IFN α and resisted CD20 has the lymphoma mantle cell cell line (Fig. 2 D) compared with hyposensitivity.Jeko-1 is only moderate for the anti-CD20MAb of parent Sensitivity, in EC₅₀Close to during 1nM with 10% maximum suppression (I_max).As shown in 734-2b, Jeko-1 (I_max=43%； EC₅₀=23pM) compared to Daudi (I_max=90%；EC₅₀=7.5pM) it is smaller to IFN α 2b responses.Compared to 734-2b, 20- 2b suppresses Jeko-1 (I in bigger degree_max=65%) and two-phase dose-response curve (Fig. 2 D) is shown.In ＜ 10pM Under, it was observed that being attributed to the low concentration response of IFN α 2b activity, it is in I_max=43% stabilization, it is similar to 734-2b.In 100pM High concentration response, wherein I can be substantially observed above_maxReach 65%.20-2b low concentration IFN α 2b responses (EC₅₀= It is 0.97pM) 25 times more effective than 734-2b, similar with using Daudi result.

Combination to parent's anti-CD 20 antibodies and 734-2b (dimension trastuzumab+734-2b) is measured to illustrate 20-2b Whether the increase of effect is attributed to CD20 and IFN α signal transduction plus and/cooperative effect.V-mab+734-2b dose response Curve is most of similar to single 734-2b, except in ＞ 1nM, the suppression reinforcement of the former rather than the latter.These result tables Bright MAb targetings cause 20-2b EC₅₀It is relatively low, and its larger I_maxObviously it is attributed to adding for IFN α 2b and CD-20 signal transduction And activity.The effect of CD20 signal transductions only responds (EC in 20-2b high concentration₅₀=0.85nM) in it is obvious, its with to v-mab (EC₅₀=1.5nM) response it is parallel.V-mab+734-2b two-phase dose-response curve unobvious, because the two response weights It is folded.However, there is obvious additive effect under ＞ 1nM concentration.20-2b I_max(65%) than IFN α 2b (I_max=43%) and Tie up trastuzumab (I_max=10%) add the response of sum high, show possibility between the effect of IFN α 2b and v-mab (dimension trastuzumab) Cooperative effect be present.

ADCC activity

IFN α can strengthen ADCC activity by activated NK and macrophage, and it is basic for anti-CD20 immunotherapies Mechanism of action (MOA).We use PMBC (PBMC) to be compared as effector cell under two kinds of NHL cell lines 20-2b and v-mab ADCC.Using the replication method of the PBMC from multiple contributors as one man show 20-2b compared to V-mab has the ADCC of enhancing, as shown on Daudi and Raji cells (Fig. 4 A).It is (a kind of that the effect is also manifested in 22-2b Include the MAb-IFN α of anti-CD22MAb epratuzumabs), and the medium ADCC of its display (Carnahan etc., 2007, Mol Immunol44：1331-41).

CDC activity

CDC is considered as the important MOA of the anti-CD20MAb of I types (including v-mab and Rituximab).However, with Tosi Monoclonal antibody is not to lack this function (Cardarelli etc., 2002, Cancer Immunol in the II types MAb of representative Immunother51：15-24), although it still has anti-lymphadenoma activity.Unlike v-mab, 20-2b is in vitro not Show that CDC is active (Fig. 4 B).These results are based on C with other_HThe DNL structures of 3-AD2-IgG-v-mab modules are consistent, and it can It can be disturbed by steric hindrance, make wherein complement fixation by obvious infringement (Rossi etc., 2008, Cancer Res68：8384-92).

Embodiment 7.20-2b pharmacokinetics (PK) analysis.

In Male Swiss-Webster mice evaluate 20-2b pharmacokinetics (PK) property and with PEGASYS, PEG- intrones and α 2b-413 (the Pegylation IFN prepared using DNL, referring to U.S. Patent Application Serial No.11/925, 408) it is compared.It is dense in blood serum sample that IFN-α is determined under multiple times according to the explanation of manufacturer using ELISA Degree.In brief, blood serum sample is suitably diluted according to the mankind's IFN-α reference material provided in kit.It is incorporated into micro drop The antibody capture interferon in fixed board hole.Then the interferon combined using secondary antibody display, tetramethyl benzidine is being added (TMB) after, the quantitative interferon of the conjugated anti-secondary antibody to horseradish peroxidase (HRP) is passed through.Read plate at 450 nm.Fig. 3 The result of PK analyses is presented, it shows 20-2b compared to other agent significantly slower elimination and longer plasma residence phase.Noting In the case of dosage is penetrated as 210pmol, the pharmacokinetics serum half-life (in hours) for calculating gained is 8.0 hours (20- 2b), 5.7 hours (α 2b-413), 4.7 hours (PEGASYS) and 2.6 hours (PEG- intrones).Supersession rate (1/h) is 0.087 (20-2b), 0.121 (α 2b-413), 0.149 (PEGASYS) and 0.265 (PEG- intrones).Calculate gained MRT_0.08→ ∞ (hour) is 22.2 (20-2b), 12.5 (α 2b-413), 10.7 (PEGASYS) and 6.0 (PEG- intrones).By More it is decided by the property rather than single antibody or cell factor of the compound in pharmacokinetic parameter, it is expected that carefully The PK characteristics of intracellular cytokine-DNL compounds can be universally used in other cytokine moieties and antibody moiety and be not limited to be discussed above Specific 20-2b constructs.

It is active inside embodiment 8.20-2b

Serum stability

20-2b is in 37 DEG C of stable (not shown) in human serum (>=10 days) or whole blood (>=6 days).Use bispecific ELISA determination methods determine the concentration of 20-2b compounds.In the period of determination method, serum 20-2b is horizontal in whole blood or serum It there is no detectable change.

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

We compare 20-2b, v-mab, 734-2b or v-mab+734-2b and leaching are eliminated from whole blood under in vitro configuration The ability (Fig. 5) of bar knurl or normal B cells.Naked anti-CD20MAb therapeutic efficiency is considered as by three kinds of mechanism of action (MOA) Realization-apoptosis or the growth retardation of signal transduction induction, ADCC and CDC (Glennie etc., 2007, Mol Immunol44： 3823-37).In the determination method, v-mab can use all three MOA, however, being based on results of in vitro studies, 20-2b can dive Using signal transduction and strengthen ADCC rather than CDC on ground.In the short-run model, 20-2b and 734-2b IFN α 2b groups can Tumour cell is directly acted on, strengthens v-mab ADCC activity, and there may be some immunostimulations.However, continuing In the ex vivo assays of two days, the spectrum entirely for the IFN α mediation for being not carried out in vivo occurring is congenital and adaptive immunity system The activation of system.

Under 0.01nM, 20-2b (60.5%) is than v-mab (22.8%), 734-2b (38.6%) or v-mab+734-2b (41.7%) significantly more Daudi cells (Fig. 5) are consumed.Under 0.1nM, 20-2b and the similar journey of v-mab+734-2b consumption The Daudi (88.9%) of degree, it is more (Fig. 5) than v-mab (82.4%) or 734-2b (40.7%).Under 1nM, except 734-2b (55.7%), each dose of consumption ＞ 95% Daudi (Fig. 5).Each indicated difference has statistical significance (P ＜ 0.01).

For IFN α 2b and v-mab, Ramos not as Daudi sensitivities.734-2b effect is only medium, in each concentration Under cause Ramos consumption ＜ 20% (Fig. 5).Under 0.01 and 0.1nM, 20-2b is more more than v-mab+734-2b consumption Ramos, it transfers to eliminate more cells (Fig. 5) than v-mab.Under 1nM, in addition to 734-2b all processing all obtain Similar Ramos consumption (75%) (Fig. 5).Each indicated difference has statistical significance (P ＜ 0.02).

As shown on 734-2b, independent IFN α 2b can not consume normal B cells in the determination method.In these low concentrations Under, 20-2b, v-mab and v-mab+734-2b each show similar B cell dose response consumption, its considerably less than Daudi or Ramos consumption.All processing will not result in significant t cell depletion (data are not shown).

20-2b in SCID mice inside effect

One of mouse model is limited to extremely low sensitiveness of the murine cells to mankind's IFN α 2b.20-2b can be in the mankind The wholistic therapy benefit of realization may include to strengthen congenital and adaptive immunity.After these limitations are understood, we have studied 20-2b anti-lymphadenoma effects inside SCID mice moderate resistance dissemination Burkitt lymphoma model.Originally we test height Sensitiveness early stage Daudi models (Fig. 6 A).One day after inoculation, to each group apply single low dosage 20-2b, v-mab or 734-2b.The 0.7pmol (170ng) of single dose v-mab (dimension trastuzumab) or 734-2b are compared with salt solution, v- Mab causes survival rate to significantly improve (P ＜ 0.0001), but uncorrelated MAb-IFN α control 734-2b are without this performance (Fig. 6 A). This raising is medium, and Median survival time (MST) increased to 34 days of v-mab from 27 days of salt solution.However, single dose 0.7pmol (170ng) 20-2b MST is improved more than 100 days (P ＜ 0.0001) compared to saline control and v-mab groups (Fig. 6 A).The research was terminated after 19 weeks, and now 7 long-term survivorses (LTS) in 0.7pmol20-2b treatment groups are entered Row ptomatopsia, visible disease indication (healing) (Fig. 6 A) is not found.It is worth noting that, even if lowest dose level 0.07pmol (17ng) 20-2b not only makes MST double (Fig. 6 A).

Next, we assess the effect of 20-2b is in the late period Daudi model of more challenge, wherein making mouse exist Before processing forms generally bigger tumor load (Fig. 6 B).Seven days after tumor inoculation, to each group apply single low dosage (0.7, 7.0 or 70pmol) 20-2b, v-mab, 734-2b or PEGASYS.The MST of saline control mouse is 21 days (Fig. 6 B).Highest The PEGASYS or 734-2b of dosage (70pmol) each have the Pk (compared to restructuring IFN α 2b) of enhancing, but do not target In tumour, they make MST double (42 days；P ＜ 0.0001) (Fig. 6 B).Using 20-2b under 100 times of low dosages (0.7pmol) Processing produces the result (38.5 day) (Fig. 6 B) similar to the PEGASYS or 734-2b of maximum dose level (70pmol).Use 20-2b Processing causes the survival rate compared to PEGASYS or the 734-2b processing using 70pmol to show under 10 times of low dosages (7pmol) Write and improve (80.5 days, 20%LTS) (P ＜ 0.0012) (Fig. 6 B).Under the maximum dose level (70pmol) tested, 20-2b will MST increases to ＞ 105 days and 100%LTS (Fig. 6 B) be present.In infantile tumour model we previously verified v-mab The survival of the mouse with Daudi can be increased under relatively low dosage (3.5pmol), and higher dosage then produces LTS.So And in the late stage tumor model, single dose only has the function that notable but medium for 70pmol v-mab for survival (MST=24 days, P=0.0001) (Fig. 6 B).

We then determine 20-2b in the model of more challenge, the model for the direct rejection ratio Daudi of IFN α more It is insensitive, and be more not responding to for v-mab immunotherapies.Raji is compared to the susceptibility that Daudi directly acts on for IFN α 2b It is small about 1000 times.However, Raji have the CD20 antigen density similar with Daudi (Stein etc., 2006, Blood108：2736- 44) and to v-mab respond, but the response than Daudi significantly much smaller (Goldenberg etc., 2009, Blood113,1062- 70).Start to treat after five days in tumor inoculation, the effect of 20-2b is late studied in Raji models (Fig. 7 A).It is circumferential each through two Group applies totally 6 injections (each 250pmol).Relative to salt solution, 734-2b does not increase survival (MST=16 days), this with Raji is consistent (Fig. 7 A) to the insensitivity of IFN α.Relative to salt solution, V-mab dramatically increase survival (MST=26 days, P ＜ 0.000l) (Fig. 7 A).20-2b is more more effective (MST=33 days, P ＜ 0.0001) (Fig. 7 A) than other all processing.

Finally, we 20-2b is have studied in the case of NAMALWA the effect of (Fig. 7 B), NAMALWA is a kind of to IFN α Directly effect has hyposensitivity, about 25 times low compared to Daudi or Raji CD20 antigen densities, and is considered as resisting The resistant human lymphoma of CD20 immunotherapies (Stein etc., 2006).Totally 6 dosage (each dosage is applied to each group 20-2b or 734-2b 250pmol).The v-mab for applying totally 7 dosage (each dosage 3.5nmol) to another group.Use salt solution The group of processing has the MST (Fig. 7 B) of 17 days.The group handled with 734-2b has significantly but very the survival of moderate improves (MST=20 days, P=0.0012) (Fig. 7 B).20-2b (MST=34 days) is than 734-2b (P=0.0004) and v-mab (MST =24 days, P=0.0026) more effectively, and v-mab gives dose ratio 20-2b high 14 times (Fig. 7 B).

Conclusion

Acquired results clearly indicate that causes than any dose exclusive use of immune cell factor with anti-CD20MAb targeting IFN αs Or it is used in combination more potent and effective.The MAb target tumors of IFN α allow to more low-frequency scheme and give independent agent, lower Or the related side effect of IFN therapies is eliminated, and the effect of significantly strengthened.In addition, the IFN α targetted can induce acute swell Knurl orients immune response, and may be by arousing immunological memory to congenital and adaptive immunity multiple-effect sexual stimulus (Belardelli etc., 2002, Cytokine Growth Factor Rev12：119-34).Pass through the conjugated generation MAb- of chemistry Other groups of IFN α disclose some potential clinical benefit (Pelham etc., 1983, Cancer Immunol of such construct Immunother15：210-16；Ozzello etc., 1998, Breast Cancer Res Treat48：135-47).Include muroid IFN α and anti-HER2/neu MAb restructuring MAb-IFN α are shown in immunocompetent mouse to transgenosis (HER2/ Neu) the potent suppression of muroid B cell lymphoma, and protective adaptation immune response can also be induced according to immunological memory (Huang etc., 2007, J Immunol179：6881-88).

It is anticipated that 20-2b therapies will stimulate the local recruitment for the panimmunity cell for including NK, T4, T8 and dendritic cells And activation, cause cytotoxicity and the ADCC strengthened, and potentially can orient immunological memory by induced tumor.However, for the mankind IFN α 2b, the susceptibilitys of murine cells with respect to mankind's cell polar the earth reduce (about 4log) (Kramer etc., 1983, J Interferon Res3：425-35；Weck etc., 1981, J Gen Virol57：233-37).Therefore, in above-mentioned mouse model In In vivo study, though have, and considerably less 20-2b anti-lymphadenoma activity is attributable to IFN α 2b to mouse immune reaction Activation.On the contrary, death is mainly due to direct effects of the IFN α 2b to lymphoma cell.

We have shown that 20-2b can strengthen ADCC, and this is probably the most important MOA of anti-CD20 immunotherapies.However, Because mankind's IFN α 2b is only the very weak stimulant of Rat host immune effector cell, it is possible that will not as in the mankind that Sample realizes the ADCC of IFN α enhancing.Even if having these limitations, vivo results still show that 20-2b can be efficient anti-lymphadenoma Agent, 100 times more than v-mab or non-target tropism MAb-IFN α of effect is shown in IFN α sensitiveness Daudi models.Even in Directly act on IFN α relative insensitivity (Raji/NAMALWA) or resist the leaching of CD20 immunotherapies resistant (NAMALWA) In bar knurl model, the effect of 20-2b is still shown than v-mab or non-targeted MAb-IFN α higher.

IFN α 2b to v-mab fusion is by extending circulation time and allowing cancer target to strengthen its internal effect. Pk treatment conspicuousness is shown in Daudi models, wherein the PEGASYS removed more slowly is better than including than the PEG- comparatively fast removed Son, although the latter has higher activity specific (data are not shown).20-2b has significantly more than PEGASYS or 734-2b Big effect, show that target lymthoma by anti-CD20MAb plays decisive role for its more excellent effect and effect.Exceed to anticipate Expect, it is also very notable in the influence of targeting or even in vitro determination method.In vitro in proliferation experiment, only allow to pass by signal Lead and lymthoma is suppressed, relative to non-targeted MAb-IFN α, no matter in the case of v-mab alone or in combination, 20-2b Activity is shown under relatively low 25 times of concentration.In vitro configuration allows to be related to three kinds of all anti-CD20MOA.Even if not yet There is CDC activity, no matter in the case of alone or in combination, 20-2b more effectively consumes leaching than IFN α or v-mab from blood Bar knurl, indicate the importance of targeting.And the influence that MAb is targetted in vitro/vitro study is more or less unexpected, because MAb, effector and target cell are all limited in whole experiment.It is anticipated that in being tested inside human patientses, 20-2b By with generally bigger influence.

20-2b IFN α 2b and v-mab part obviously can add and/or act synergistically, to facilitate the effect that it strengthens Power.In-vitro multiplication determination method shows there is at least one addition, and it in vitro study by obtaining joint v-mab and 734- 2b confirms better than the result that any agent is used alone.This can be used as the v-mab of 20-2b part ADCC by increase Activity is realized when combining with 734-2b in vitro, but ADCC does not still have feature in proliferation assay in vitro, shows In the presence of other mechanism.The signal transduction with reference to caused by v-mab CD20 can strengthen IFN α signal, cause the effect of enhancing.It is optional Ground, internalization/downward of I type IFN acceptors can be prevented with a kind of v-mab (it is MAb of slowly internalization) combination, produces the longer time With effective IFN α inducement signal.

Embodiment 9. (Fab)₂- interferon-λ 1 DNL constructs show effective bioactivity to the cell targetted

General introduction and introduction

Interferon lambda 1 (IFN- λ 1) is the type iii interferon for being described as II type cytokines family members recently, on Antiviral and antitumor activity and immune system have treatment potentiality.IFN- λ, as I types IFN (comprising IFN-α and IFN-β), transduceed by JAK/STAT approach trigger signal, include activation, the phosphorylation of stat protein of JAK1 and TYK2 kinases And activation (Witte etc., 2010, Cytokine Growth Factor of the transcription complex of the gene factor 3 of IFN stimulations Rev21：237-51；Zhou etc., 2007, J Virol81：7749-58).

Main Differences between type III IFN and I types IFN are the distribution of its corresponding receptor complex.IFN-α/β leads to The I types interferon receptors for crossing two kinds of wide expressions carries out signal transduction, and this at least partly causes related to IFN-α/β therapies General toxicity (Pestka, 2007, J Biol Chem282：20047-51).By contrast, IFN- λ are by including IFN- λ acceptors The heterodimeric receptor complex of 1 (IFN- λ R1) and IL-10 acceptors 2 (IL-10R2) carries out signal transduction.And IL-10R2 is making Generally expressed in haemocyte and non-hematopoietic cell, IFN- λ R1 have height-limited expression pattern, wherein in epithelial cell, black Horizontal highest in plain cell and liver cell, and in primary central nervous system cell it is horizontal it is minimum (Wolk etc., 2005, Genes Immun6：8-18).Although blood immune cells express IFN- λ R1, it can suppress IFN- λ 1 effect due to secretion The short splice variants of IFN- λ R1 and show response to IFN- λ and be damaged (Witte etc., 2009, Genes Immun10：702- 14).The finite response of neuronal cell and immunocyte also promotes IFN- λ toxicity to be decreased compared to I types IFN (Witte etc., 2009, Genes Immun10：702-14).

IFN- λ show architectural feature as the cytokine class relevant with IL-10, but it is antiviral and anti-to show I type IFN classes Proliferation 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 IFN- λ 1 and IFN- λ 2 Virus replication or various viral cytopathic effects can be reduced, the virus includes DNA virus, such as hepatitis type B virus (Robek etc., 2005, J Virol79：3851-54；Doyle etc., 2006, Hepatology44：896-906) and the simple blister of 2 types Exanthema 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 HCV (HCV) (Robek etc., 2005, J Virol79：3851-54；Doyle etc., 2006, Hepatology44：896-906)；Adopted single strand RNA virus is born, for example, it is vesiculovirus Stomatovirus (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 double-stranded RNA Virus, such as rotavirus (Pott etc., 2011, PNAS USA108：7944-49).IFN- λ 3 are reflected according to genetic research Be set in HCV infection key cytokines (Ge etc., 2009, Nature461：399-401), and have for EMCV most Effective active (Dellgren etc., 2009, Genes Immun10：125-31).

IFN- λ antiproliferative activity, including neuroendocrine are also determined in several human cancer cell lines Cancer BON1 (Zitzmann etc., 2006, BBRC344：1334-41), glioblastoma LN319 (Meager etc., 2005, Cytokine31：109-18), immortalized keratinocytes HaCaT (Maher etc., 2008, Cancer Biol Ther7： 1109-15), melanoma F01 (Guenterberg etc., 2010, Mol Cancer Ther9：510-20) and cancer of the esophagus TE-11 (Li etc., 2010, Eur J Cancer46：180-90).In animal model, IFN- λ are anti-by congenital and adaptive immunity Inducing apoptosis of tumour cell and elimination are answered, this shows that IFN- λ local delivery is probably useful in human malignancies treatment Strategy (Numasaki etc., 2007, J Immunol178：5086-98；Sato etc., 2006, J Immunol176：7686-94).

The conjugated mankind IFN- λ 1 (PEG-IFN- λ 1) in 20-kDa polyethylene glycol are currently under chronic HCV infection treatment Clinical development in.In the Ib stages are studied, antiviral activity is observed under each dosage (0.5-3.0 μ g/kg), and When PEG-IFN- λ 1 to be applied to the 1 type HCV patient of gene recurred after IFN-α therapy, virus load is down to from 2.3 4.0log (Muir etc., 2010, Hepatology52：822-32；Ramos, 2010, J Interferon Cytokine Res30：591-95).The IIb stages study (Zeuzem etc., 2011, J Hepatology54：5538-39) report HCV patient (type of gene 1 and 4 types) have significantly higher reaction for being treated with PEG-IFN- λ 1 compared to the treatment of PEG- Intederon Alpha-2as Rate, and using PEG-IFN- λ 1 compared to other advantages using PEG- Intederon Alpha-2as include adverse events frequency it is relatively low, The frequency of influenza-like symptom, anaemia and musculoskeletal symptom reduces, and seldom observes Neutrophilic granulocytopenia and blood platelet Reduce.However, compared with PEG- Intederon Alpha-2as, the hepatotoxicity wind agitation of visible more height ratio in maximum dose level PEG-IFN- λ 1 (Zeuzem etc., 2011, J Hepatology54：5538-39).In order to improve IFN- λ 1 effect and security overview, we Construct module DOCK-AND-LOCK^TM(DNL^TM) compound come tie this cell factor with Fab albumen stabilisation two The targeting antibodies of dimer form.

Humanized antibody hRS7 (anti-Trop2), hMN15 (anti-CEACAM6), hL243 (anti-HLA-DR) or c225 will be derived from The Fab-DDD2 modules dimerization of (inosculating antibody EGFT) and be connected with AD2-IFN- λ 1 with produce respectively immune cell factor (E1)- λ 1, (15)-λ 1, (C2)-λ 1 and (c225)-λ 1.It will be appreciated by those of ordinary skill in the art that the present invention be not limited to it is exemplary Antibody, but can be implemented using any known antibodies.Evaluate the bioactivity of interferon-antibody construct and with targetting carefully Born of the same parents are to be compared with the recombinant human IFN- λ 1 (rhIFN- λ 1) in non-targeted cell line.It is observed that in (E1)-λ 1 HRS7Fab dimers make IFN- λ 1 significantly increase (about 1000 times) for the vitro efficacy of several Trop2- positive cell lines, The cell line includes human cervical cancer ME-180 (EC₅₀＜ 0.1pM), lung squamous cancer SK-MES-1 (maximum 60% suppress) and food Road cancer TE-11 (maximum 45% suppresses).Similarly, in CEACAM6- positive ME-180 cells (EC₅₀＜ 1pM) and TE-11 cells In, the growth inhibition that hMN15Fab dimers mediate IFN- λ 1 significantly increases (about 100 times), but in CEACAM6 feminine genders SK- It is really not so in MES-1 cells.

In order to study (Fab)₂- IFN- λ 1 antiviral activity, we compare rhIFN- λ 1, (15)-λ 1 and (C2)-λ 1 The influence infected and replicated in expression CEACAM6 rather than HLA-DR A549 cells for encephalomyocarditis virus (EMCV).Knot Fruit shows, rhIFN- λ 1 and (Fab)₂- IFN- λ 1 can effectively prevent the cytopathic effect of EMCV inductions, but (15)-λ 1 The antiviral efficacy of 1 high 6 times and high 10 times than (C2)-λ 1 than rhIFN- λ of display.Other studies have shown thats are thin what is targetted In born of the same parents, (Fab)₂- IFN- λ 1 can strengthen phosphorylation (the Jak-STAT letters of signal transducer and transcription activator (STAT) 1,2 and 3 Critical event in the activation of number transduction), and the cell surface expression of I classes MHC (MHC I), its Antigen is promoted to present.These data collectively show thats, the cell surface for the IFN- λ 1 that Fab antibody fragment is mediated is combined by targetting Immobilization can significantly increase its effect in the cell targetted and improve internal security overview.Those skilled in the art It will be recognized that these effects are not limited to Fab antibody fragment, but there is also in complete antibody or other antibody fragments.This A little results are shown, include the targeting antibodies for being connected to IFN- λ 1 or the DNL of antibody fragment^TMCompound is used as therapeutic agent to control Treat cancer, infectious diseases, asthma or multiple sclerosis.

Material and method

Antibody and reagent- humanized antibody hA20-IgG (anti-CD20), hRS7-IgG (anti-Trop-2), hMN15-IgG are (anti- CEACAM6) and hL243 (anti-HLA-DR) is to come from Immunomedics, Inc.Recombinant human IFN- λ 1, for mankind IFN- λ 1 Mouse mAb and be available from R＆D Systems Inc for human HLA-ABC mouse FITC-IgG1k.It is prominent comprising being connected to The amino acid sequence of the fusion protein of variant (C171S) or wild type IFN λ 1 AD2 parts and poly- His sequences is in being illustrated below. Cell Signaling Technology are available from for STAT1, STAT3, pY-STAT1 and pY-STAT3 rabbit antibody Inc.STAT2 antibody is to come from Santa Cruz Biotechnology.PY-STAT2 antibody is to come from Millipore Corporation。

Wild type：AD2-IFN-λ1

MCGQIEYLAKQIVDNAIQQAGCEFPKPSTPPGSSGGAPAMDGPVPTSKPTTTGKGCHIGRFKSLSPQELASFKKARD ALEESLKLKNWSCSSPVFPGNWDLRLLQVRERPVALEAELALTLKVLEAAAGPALEDVLDQPLHTLHHILSQLQACI QPQPTAGPRPRGRLHHWLHRLQEAPKKESAGCLEASVTFNLFRLLTRDLKYVADGNLCLRTSTHPESTVEHHHHHH (SEQ ID NQ：96)

Mutant：AD2-IFN-λ1-C171S

MCGQIEYLAKQIVDNAIQQAGCEFPKPSTPPGSSGGAPAMDGPVPTSKPTTTGKGCHIGRFKSLSPQELASFKKARD ALEESLKLKNWSCSSPVFPGNWDLRLLQVRERPVALEAELALTLKVLEAAAGPALEDVLDQPLHTLHHILSQLQACI QPQPTAGPRPRGRLHHWLHRLQEAPKKESAGCLEASVTFNLFRLLTRDLKYVADGNLSLRTSTHPESTVEHHHHHH (SEQ ID NO：97)

AD2-IFN- λ 1 expression and purifying- in order to produce it is used for DNL^TMThe modules of IFN- λ 1 that compound is formed, synthesis bag Synthetic DNA sequence (figure containing AD2, knuckle joint and the mankind INF- λ 1 (C171S) to optimize in expression in escherichia coli 8A) and it is cloned into Msc I and the Xho I sites of pET-26b carriers.The Rosetta-pLysS cells converted with plasmid (Novagen) the Difco 2xYT meat soups of 100 μ g/ml kanamycin sulfates and 34 μ g/ml chloramphenicol are supplemented with 37 DEG C Grown in (Becton Dickinson).When cell density reaches OD₆₀₀For 1.0 when, add IPTG to 0.5mM and at 30 DEG C Inducible protein expression continues 4 hours.By the way that cell and the freeze overnight at -80 DEG C is collected by centrifugation.Agglomerate is thawed and molten Solve buffer solution (2%Triton-X100,5mM MgSO₄, 10 units per ml nucleases (benzonase) (Novagen), 100 μM AEBSF, 20mM Tris-HCl, pH8.0) in homogenize.Homogenate centrifuges 30 minutes under 18,000RPM and agglomerate exists Homogenized again in PBS/1%Triton X-100, then form agglomerate again.By agglomerate be dissolved in 20ml 6M guanidine hydrochlorides, In 100mM sodium phosphates (pH8.0) and it is loaded on His-Select affinity columns (GE-Healthcare), then with 6M guanidines, 50mM Sodium phosphate (pH8.0) washs.In 4M guanidine hydrochlorides, 100mM NaH₂PO₄(pH4.5) elution protein in, and by adding 200 μ L 3M Tris-HCl (pH8.6) are neutralized, and are added DTE to 60mM and solution is kept at room temperature overnight.By the change of reduction Property protein solution rapid dilution is in 0.5M arginine, 20mM oxidizeds form of glutathione, 2mM EDTA, 100mM Tris (pH8.0) in, then for 5L renaturation buffers (0.5M arginine, 2mM oxidizeds form of glutathione, 0.6mM DTE, 2mM EDTA, 20mM Tris (pH8.0)) dialysed and kept for 72 hours at 4 DEG C.Then PBS-AG-2 buffer solutions are directed to (35.2mM Na₂PO₄.7H₂O；0.4M NaCl；6.5mM NaH₂PO₄.H₂O；150mM arginine；150mM glutamic acid, pH8.0) Dialysis solution.Final product is concentrated into about 0.5mg/ml and analyzed using SDS-PAGE.

DNL^TMConstruct- shape merged with the modules of AD2-IFN- λ 1 by hRS7-, hMN15- or hL243-Fab-DDD2 module Into DNL^TMCompound generates (Fab)₂The conjugates of-IFN- λ 1, including (E1)-λ 1, (15)-λ 1 and (C2)-λ 1 (Fig. 8 B), such as first It is preceding it is described (see, for example, United States Patent (USP) 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 is each Embodiment part be incorporated herein by reference).According to the sequence (accession number from DrugBank：DB00002) come Build c225-Fab-DDD2 modules.Sequentially purified product and SDS- is utilized on Kappa-select and His-select posts PAGE is analyzed under reduction and non reducing conditions using 4-20%Tris- glycine gels (not shown).

Cell culture and surface combine- TE-11 is carried by Hiroshi doctors Nakagawa (University of Pennsylvania) friendship For.Huh-7 and T.Tn is available from Japanology living resources collection (Japanese Collection of Research Bioresources).Every other cell line is available from American type culture collection.ME-180, TE-11 and A375 are thin Born of the same parents grow in the RPMI1640 culture mediums (Invitrogen) containing 10%FBS (Hyclone).HepG2, Huh-7 and SK-MES- 1 cell grows in the EMEM culture mediums (ATCC) containing 10%FBS.A549 is in the F12 culture mediums containing 10%FBS (InVitrogen) grown in, and T.Tn grows in the DMEM/F12 culture mediums (InVitrogen) containing 10%FBS.It is all Cell line is all in 5%CO at 37 DEG C₂Humid atmosphere in cultivate.

For binding assay, of short duration Trypsin Induced is carried out to cell, is suspended in fresh culture and is formed Agglomerate, then with 10 μ g/ml humanizations mAb or in 1%BSA-PBS, the agent based on IFN- λ 1 of serial dilution suspends again. After being incubated 45 minutes at 4 DEG C, cell is formed into agglomerate and washed twice with 1%BSA-PBS, marked at 4 DEG C with FITC Goat anti human's class IgG-Fc or mouse anti human class IFN- λ 1 is incubated 45 minutes together, the goat anti-mouse then marked with FITC IgG-Fc is detected.After washing three times, measured and combined using flow cytometry.In order to study the change of MHC I expression, Cell is set to continue 3 days exposed to 1 dose of IFN- λ, and by being tied with the mouse IgG 1k of the FITC marks for human HLA-ABC Close to detect its surface MHC1.Unspecific mouse IgG1k using FITC marks is used as negative control.

In-vitro multiplication- be seeded in ME-180, SK-MES-1, TE-11 and T.Tn cell with the density of 1000 cells/wells It is incubated overnight in 96 orifice plates and at 37 DEG C, is then exposed to 1 dose of the IFN- λ of progressive concentration, continues 4 days.Use CellTiter96 Cell Proliferation assays (Promega) determine viable cell density.

Antiviral determination method- (be referred to as using the stable Huh-7 cell lines containing HCV genotype 1 b Con1 replicons Huh-7-Con1), IFN- λ 1 and 2 are measured using HD Biosciences (China) Co., Ltd (Shanghai, China) (Fab)-λ 1 anti-HCV activity.Firefly luciferase gene is incorporated into this replicon as the report of virus levels Gene.The determination method includes three kinds of 1 dose of IFN- λ, (c225)-λ 1, (C2)-λ 1 and rhIFN- λ 1.Wherein, (c225)-λ 1 is wrapped Two Fab of chimeric mAb containing the EGFR on selectively targeted Huh-7 cell surfaces, and (C2)-λ 1 and rhIFN- λ 1 are two Non-targeted tester.Huh-7-Con1 cells are handled 3 days with these three agent, and determine disease by measuring uciferase activity Malicious levels of replication.Meanwhile also to have rated these agent using CellTiter Glo kits (Promega) thin for parent Huh-7 The cytotoxicity of born of the same parents.

It is thin using being carried out using PBL Interferon Source (Piscataway, NJ) in another determination method The effect of born of the same parents' lesion suppresses the antiviral activity that determination method measures (15)-λ 1 on the A549 cells with EMCV.In the determination method Including hMN-15-Fab-DDD2 as negative control, the reference materials of rhIFN- λ 1 (PBL Interferon Source) are as positive Control, and non-targeted controls of (the C2)-λ 1 as structure homologue.

Western blot (Western blot)- by ME-180, HepG2 and A375 cell with 5 × 10⁵Individual cells/well Density applies and is laid in 6 orifice plates and is incubated overnight at 37 DEG C.In order to evaluate STAT activation, with 1 dose of the shown IFN- λ of decreasing concentration Handle cell 1 hour, then use PhosphoSafe^TMExtracts reagent (EMD) dissolves.Cell dissolving production is isolated on SDS-PAGE Thing, it is transferred in nitrocellulose membrane (Bio-Rad), then with the rabbit antibody and phosphoric acid for total STAT1, STAT2 or STAT3 Tyrosine-specific antibody pY-STAT1, pY-STAT2 or pY-STAT3 produce trace, are detected with HRP- goat anti-rabbit antibodies.Will Beta-actin antibody is used as loading control.

It is subcutaneously injected 2.4nmol's to the female athymic nude mouse (Taconic, Germantown, NY) of four group of 8 week old (E1)-λ 1 and 6 hours, 16 hours, 24 hours and 48 hours take a blood sample.Measured using enzyme-linked immunosorbent assay (ELISA) Completely (E1)-λ 1 serum-concentration.Using the non-compartment analysis programs of WinNonLin (5.3 editions, Pharsight Corporation, St.Louis, MO) calculate pharmacokinetic parameter.It is also thin by ME-180 in order to confirm ELISA result The vitro proliferation determination method of born of the same parents, use (E1)-λ 1 to be used as reference material, have rated biologies of the IFN- λ 1 in selective blood serum sample Activity.

RT-PCR is analyzed.With 1 dose of IFN- λ processing HepG2 cells 24 hours and useReagent (Life Technologies total serum IgE) is separated.UseIII single step RT-PCR systems (Life Technologies) profit The mRNA for analyzing myxovirus resistance A (MxA) gene under the following conditions with forward and reverse primer is expressed：- 55 DEG C of cDNA synthesis/ Continue within 30 minutes a circulation and continue within PCR-94 DEG C/15 seconds, 62 DEG C/30 seconds, 68 DEG C/30 seconds 25 circulations.With inside Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA 452-bp fragments are expanded under conditions of control is similar.

Pharmacokinetics in mouseTo four group of 8 week old female athymic nude mouse (Taconic, Germantown, NY 2.4nmol (E1)-λ 1) is subcutaneously injected and was taken a blood sample at 6 hours, 16 hours, 24 hours and 48 hours.Inhaled using enzyme linked immunological Attached complete (E1)-λ 1 of determination method (ELISA) measurement serum-concentration.Using the non-compartment analysis programs of WinNonLin (5.3 editions, Pharsight Corporation, St.Louis, MO) calculate pharmacokinetic parameter.It is also logical in order to confirm ELISA result The vitro proliferation determination method of ME-180 cells is crossed, uses (E1)-λ 1 to be used as reference material, have rated IFN- λ 1 in selective serum sample Bioactivity in product.

Statistical analysis.Use Prism GraphPad software kits (Advanced Graphics Software, Rancho Santa Fe, CA) with F examine the resultful statistical significance (P ＜ 0.05) of measure institute.

As a result

(Fab)₂- IFN- λ 1 generation and sign- point mutation (C171S) is introduced into the sequences of wild type IFN- λ 1 to eliminate The potential interference of the unpaired cysteine residues of refolding and assembling with DNL modules.AD2- is produced in Escherichia coli The recombinant forms of the modules of IFN- λ 1 and by immobilized metal affinity chromatography (IMAC) under Denaturing from inclusion body Middle purifying.Crude protein is gone back with dithiothreitol (DTT) (DTE), then in the refolding buffer solution containing oxidized form of glutathione again Fold to form disulfide bond to allow.As determined using SDS-PAGE (not shown), the protein of AD2-IFN- λ 1 is highly purified And mainly exist with free state.The yield of final product is about 6mg/ml Bacillus coli cells cultures.

As seen in fig. 8b, by the way that the modules of AD2-IFN- λ 1 and Fab-DDD2 block combiners are generated (Fab)₂-IFN-λ 1 conjugates.In current research, by hRS7, hMN15 or hL243 Fab-DDD2 modules and the AD2-IFN- λ of molar excess 1 mixing, and be incubated overnight with 1mmol/L reduced glutathione, then add oxidized form of glutathione (2mmol/L). Using Kappa-select post purification reaction mixtures, and successfully generate four kinds of conjugates (E1)-λ 1, (15)-λ 1, (C2)-λ 1 and (c225)-λ 1.The purity of these conjugates is shown using SDS-PAGE, it isolates three in reducibility gel Bands of a spectrum (Fab DDD2- heavy chains, Fab light chains and AD2-IFN- λ 1) and the isolation essential high molecular amount band in non-reducing gel (not shown).

The cell surface expression of antigen- pass through flow cytometry measure Trop-2, CEACAM6, HLA-DR and EGFR In seven kinds of human cancer cell lines (cervix cancer, ME-180；Cancer of the esophagus, TE-11；Lung cancer, A549, SK-MES-1；Liver cancer, HepG2、Huh-7；With melanoma-cutaneum carcinoma, A375) cell surface on expression.As shown in Figure 9, Trop-2 Altimeter is reached on ME-180 and TE-11 cells, and moderate is expressed on SK-MES-1 cells.Except A375 and SK-MES-1 Outside, CEACAM6 is expressed in every other cell line, wherein the horizontal highest on A549 and HelpG2 cells.A375 is thin Born of the same parents are the high expression for showing HLA-DR.It was observed that high tables of the EGFR for Huh-7, ME-180, TE-11, SK-MES-1 and A549 Reach.These data provide (Fab)₂The basis of the further research of-IFN- λ 1 cell-specific targeting.

(Fab)₂The immobilization of the enhancings of-IFN- λ 1 on cell surface- compared with the modules of AD2-IFN- λ 1, (Fab)₂- IFN- λ 1 are shown to be significantly increased with the institute cytotropic binding affinity of target.Under 8nM concentration, such as by mouse anti human class IFN- λ 1mAb is detected, and compared to the modules of AD2-IFN- λ 1, (E1)-λ 1 is high 107 times (Figure 10 A) for ME-180 binding signal, (15)-λ 1 is high 15 times (Figure 10 B) for HepG2 binding signal, and (C2)-λ 1 is high for the binding signal of A375 cells 508 times (Figure 10 C).These data are shown and (Fab)₂Conjugated IFN- λ 1 are presented on targetted cell surface and significantly increased Immobilization.

Growth in vitro suppresses and cytotoxicity- identify that cervix cancer ME-180 is to 1 extremely sensitive cell lines of IFN- λ. RhIFN- λ 1 almost completely inhibit cell growth, and AD under the concentration higher than 10nM₂The modules of-IFN- λ 1 are shown and rhIFN- Activity (EC equal λ 1₅₀=0.1nM, data are not shown).Spies of the IFN- λ 1 for ME-180 in conjugates (E1)-λ 1 is targetted Specific activities significantly increase (EC₅₀＜ 0.1pM), its combination than AD2-IFN- λ 1 or AD2-IFN- λ 1 and AD2-hRS7-Fab High more than 1000 times (Figure 11).Compared with ME-180, the sensitivity of lung squamous cancer SK-MES-1 and cancer of the esophagus TE-11 cells to IFN- λ 1 Property smaller and IFN- λ 1 in maximum concentration under only display be respectively maximum 60% and 45% growth inhibition (not shown).So And in both cell lines still observe (E1)-λ 1 enhancing activity, than AD2-IFN- λ 1 or AD2-IFN- λ 1 with The AD2-hRS7-Fab high about 1000 times of (not shown) of combination.In addition, we have rated (15)-λ 1 for the CEACAM6- positives The activity of ME-180 and TE-11 cell lines.As shown in Figure 12, (15)-λ 1 shows than AD2-IFN- λ 1 or AD2-IFN- λ 1 With the hM [activity of N15-IgG-AD2 high 100 times of combination.(15) the ECs of-λ 1 for ME-180₅₀It is about 1pM, it is than (E1)-λ 1 It is high 10 times.

Antiviral activity- measured respectively in Huh-7 and A549 cells selectivity the constructs of 2 (Fab)-λ 1 be directed to HCV With EMCV antiviral activity.In expression EGFR Huh-7 cells, when suppressing HCV duplications, (the EC of (c225)-λ 1₅₀= 0.56pM) than (EC of non-target tropism (C2)-λ 1₅₀=91.2pM) and (EC of business rhIFN- λ 1₅₀=69.2pM) it is more effective respectively 163 times and 123 times (Figure 13 A).In expression CEACAM6 A549 cells, (15)-λ 1 is compared to the Hes of non-target tropism (C2)-λ 1 RhIFN- λ 1 show that high 10 times and 6 times of anti-EMCV is active (Figure 13 B) respectively.It is worth noting that, (C2)-λ 1 is remained RhIFN- λ 1 65% to 75% antiviral activity.These results instruction IFN- λ 1 cell surface targeting can effectively strengthen it Antiviral activity.

Cellular signal transduction- in order to understand (Fab)₂Strengthen the mechanism of the activity of IFN- λ 1, we compare (Fab)₂-IFN-λ 1 and AD2-IFN-, 1 signaling activities in three kinds of cell line of λ.Phosphorylation assays show that 15-Fab-DDD2 does not exist Any STAT phosphorylations are induced in HepG2 cells, and AD2-IFN- λ 1 induce detectable phosphorylation (not under 0.1nM concentration Show).By contrast, (15)-λ 1 induces three kinds of STAT, particularly STAT1 notable bigger phosphorylation, shows 0.01nM (15)-λ 1 signaling activity is no better than the (not shown) of 0.1nMAD2-IFN- λ 1.(E1)-λ 1 target ME-180 and (C2) also show (Fab) in the A375 cells that-λ 1 is targetted₂- IFN- λ 1 strengthen relative to AD2-IFN- λ 1 STAT phosphorylations (data are not shown).

(15)-λ 1 also have studied by flow cytometry MHCI classes antigen (MHC-I) is raised in HepG2 cells The ability (not shown) of cell surface expression.And continue three days with most 1nM hMN-15-Fab-DDD2 processing HepG2 cells Make MHC-1 surface level unchanged (MFI about 50), MFI increases by 3 are observed in the cell that as little as 1pM (15)-λ 1 is handled More than times (about 170).Under 100pM (15)-λ 1, MHC-1 expression reaches maximum horizontal (MFI about 270).Although AD2- IFN- λ 1 can raise MHC-1, but it needs higher concentration (100pM) just effective (not shown).

We have also investigated (15)-λ 1 to induce myxovirus resistance A (MxA) gene (fudiciary marker of IFN bioactivity) Expression ability, and utilize RT-PCR measurement results.Untreated or thin by the HepG2 of hMN-15-Fab-DDD2 processing In born of the same parents, fail to detect MxA mRNA (not shown) using RT-PCR.By 0.1pM (15)-λ 1 or 10pM AD2-IFN- λ 1 The inducing action of MxA genes is obvious (not shown) in the cell of processing, therefore further demonstrates (15)-λ 1 and be better than AD2- IFN- λ 1, particularly general 2 (Fab)-λ 1 are better than IFN- λ 1 advantage.

Pharmacokinetics in mouse.After single dose (2.4nmol) subcutaneous administration, complete (E1)-λ's 1 is averaged Serum-concentration reached high level at 6 hours and the following (not shown) of ELISA detection limits was down at 48 hours.From non- Pharmacokinetics (PK) parameter of compartment analysis shows that mean residence time is 12 hours, T_1/2For 8.6 hours, and remove speed Rate is 2.2ml/h.When measuring it in blood for the inhibitory activity of ME-180 cells also by (E1)-λ 1 using MTS determination methods During concentration in final proof product, as a result largely consistent (not shown).

Restructuring IFN-α has very quick clearance rate in mouse, and display mean residence time is only 0.7 hour. Therefore, 2 (Fab)-λ 1 show the PK (not shown) that significantly improves suitable with PEG-IFN- α [37] and PEG-IFN- λ.

Discuss

Type iii interferon (IFN) comprising IFN- λ 1, IFN- λ 2 and IFN- λ 3 cause it is antiviral, antitumor and immune Regulation activity aspect is similar with IFN-α behavior.Due to its more limited cell target, IFN- λ are for as showing based on IFN-α There is the potential substitute of therapeutic scheme attractive.By the way that hRS7 (the anti-Trop-2 of humanization), hMN-15 (humanizations will be derived from Anti- CEACAM6), hL243 (the anti-HLA-DR of humanization) and c225 (inosculating antibody EGFR) stabilisation Fab dimers and IFN- λ 1 Point specifically ties together, forms the Novel free for being known respectively as (E1)-λ 1, (15)-λ 1, (C2)-λ 1 and (c225)-λ 1 Epidemic disease cell factor, we are prepared for including the conjugated IFN- λ 1 of antibody DOCK-AND-LOCK^TMCompound is to improve IFN- λ 1 in institute Antiproliferative effect (at most 1,000 times) in the cancer cell system of targeting.Compared with non-target tropism (C2)-λ 1, pass through (15)-λ 1 Or the targeted deliveries of IFN- λ 1 to respective antigen-expressing cells are also added significantly antiviral activity by (c225)-λ 1, are such as directed to (15)-λ 1 of encephalomyocarditis virus (EC in mankind's adenocarcinoma of lung epithelial cell line A549₅₀=22.2pM is to 223pM) and be directed to (c225)-λ 1 of HCV (EC in Polymerase chain reaction Huh-7₅₀=0.56pM is to 91.2pM) shown in.These It is astonishing and unexpected result is that be attributed to the more preferable positioning for the cell that IFN- λ 1 are targetted to antibody and stronger combination, And favourable pharmacokinetic profile (Ts of (the E1)-λ 1 in mouse_1/2=8.6 hours).

Trop-2 and CEACAM6 is with the horizontal expression higher compared to IFN- λ 1 acceptor in (not showing on target cell Go out).Therefore, the molecules of IFN- λ 1 more than 10 times can be incorporated into target cell in the form of DNL conjugates.Trop-2 or CEACAM6 with The common connection of IFN- λ 1 heterodimeric acceptor can also make IFN- λ 1 almost 100 times of bond strength increase, and such as (E1)-λ 1 exists Shown in ME-180.Immune conjugated IFN- λ 1 targeted delivery is compared to the antibody and interference respectively applied alone or in combination Element provides the significantly bigger bioactivity treated for tumour and infectious diseases.

IFN- λ are explored as the potentiality of the treatment substitute of IFN-α by the use of PEG-IFN- λ 1, and it is in clinical studies Show security overview (Miller etc., 2009, Ann N Y Acad to be increased compared to PEG-IFN- α -2a Sci1182：80-87；Ramos, 2010, J Interferon Cytokine Res30：591-95).However, for PEG- The patient of IFN- λ 1 and PEG-IFN- α -2a processing, the incidence of some serious adverse events (including dose-limiting hepatotoxicity wind agitation) It is similar, and even frequent in the patient handled with the PEG-IFN- λ 1 of maximum dose level (Zeuzem etc., 2011, J Hepatology54：5538-38).On the other hand, for some cancers (Meager etc., 2005, Cytokine31：109-18) Or virus (Ank etc., 2006, J Interferon Cytokine Res26：373-79), it is effective to be not so good as I types IFN by IFN- λ.I Assume the surface antigen that IFN- λ are connected to for great expression having specific antibody and can strengthen it and determine in target cell Position, so as to produce bigger effect and be hopeful make it that the toxicity for non-target cell is smaller.Therefore, we have developed four The prototype of immune cell factor of the kind based on IFN- λ, the conjugated stabilisation dimer in Fab of each self-contained locus specificity IFN- λ 1, and show its superiority compared to not conjugated parent's module alone or in combination.In antitumor and antiviral survey Determine to show improved effect in method, and this with can be combined by the cell surface for the enhancing that composition antibody is realized and signal turn Lead consistent.

In antitumor research, we determined that ME-180, as the cell line most sensitive to IFN- λ 1, it has 80% Maximum suppression effect above and EC₅₀Low compared to other cell lines reported in document 20 times (Zitzman etc., 2006, BBRC344：1334-41；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, Trop-2 is in ME-180 On great expression make it that these tumour cells are extremely sensitive to (E1)-λ 1, under 1fM can detect growth inhibition effect and EC₅₀(＜ 0.1pM) is compared to (the EC of AD2-IFN- λ 1₅₀About 100pM) it is low 1000 times.In TE-11, SK-MES-1 and other cancers It was additionally observed that suppressing the similar enhancing of effect in cell line.Because IFN- λ also induce the reaction of congenital and adaptive immunity, this Do not evaluated herein, and in view of display IFN- λ including B16 melanomas, BNL hepatomas and MCA205 fiber meat The recent research of constructive expression in the several muroid cancer cell system of knurl, although lacking internal antiproliferative activity, lead to Cross and raise immunocyte and relevant cell factor and significantly suppress the growth and metastasis of tumours in homogenic mouse model (Numasaki etc., 2007, J Immunol178：5086-98；Abushahba etc., 2010, Cancer Immunol Immunother59：1059-71：Lasfar etc., 2006, Cancer Res66：4468-77), it is assumed that IFN- λ targeting Delivering will be similar to that constructive expressions of the IFN- λ in cancer cell, so as to produce local immunity reaction in immunotherapy for cancer With cytotoxicity (Pardoll and Drake, 2012, the J Exp Med209 of enhancing：201-9).

In antiviral study, (the c225)-λ 1 and EGFR- positives Huh-7 based on HCV genotype 1 b Con1 replicons The selectively targeted of cell shows that antiviral efficacy strengthens 123 times respectively compared to non-target tropism rhIFN- λ 1 and (C2)-λ 1 With 163 times.In another determination method, (15)-λ 1 is targetted to the CEACAM6- positive A549 cells excited with EMCV and shown 6 times and 10 times are respectively increased compared to non-target tropism rhIFN- λ 1 and (C2)-λ 1 in antiviral protection.(c225)-λ 1 and (15)-λ 1 Between effect difference be probably by cell/virus system that it is targetted is different to IFN- λ 1 sensitiveness.Previous In research (Marcello etc., 2006, Gastroenterol131：1887-98), rhIFN- λ 1 compare in Huh-7/HCV systems RhIFN- α effect is low 10 times, but in A549EMCV effect it is low 210 times (Meager etc., 2005, Cytokine31：109-18). Although it is high that the antiviral activity enhancing that (15)-λ 1 is induced in target cell is not so good as (c225)-λ 1, Pegylation is considered IFN only retains non-Pegylation IFN about less than 30% activity, this be still one it is significant find (Grace etc., 2005, J Biol Chem280：6327-36), and AD2-IFN- λ 1 activity specific is similar with rhIFN- λ 1.Therefore, 2 (Fab)-λ 1 can allow relatively low compared to PEG-IFN- λ 1 used in current clinical research dosage and frequency is identical or smaller Dosage regimen.

As therapeutic agent, restructuring IFN is limited by its very quick clearance rate.Based on previous research, restructuring IFN-α -2b, PEG-IFN- α -2a and PEG-IFN- α -2b show the half-life period of 0.7 hour, 14.9 hours and 9.3 hours respectively (Rossi etc., 2009, Blood114：3864-71).Therefore, 1 half-life period suitable with PEG-IFN- α in mouse of (E1)-λ (8.6 hours) are promising for interior therapeutic use.

Embodiment 10. is used for (Fab) for treating Alzheimer's₂- interferon-λ 1 DNL constructs

Alzheimer's (AD) is that a kind of Clinical symptoms is that progressive hypomnesia, confusion, body are gradual Deteriorate simultaneously final dead degenerative brain illness.There are about 15,000,000 people to be invaded by Alzheimer's in worldwide Disturb.Histologically, the feature of the disease is neuritic plaques, is primarily present in association's cortex, limbic system and basal ganglia In section.The main component of these patches is amyloid beta peptide (A β), and it splits for amyloid beta-protein precursor (β APP's or APP) Solve product.

A β seem there is key effect in the neuropathology of Alzheimer's.The familial form of the disease with APP mutation and early ageing gene it is relevant (Tanzi etc., 1996, Neurobiol.Dis.3：159-168；Hardy, 1996, Ann.Med.28：255-258).Disease related mutation in these genes causes the A β of 42 amino acid forms generation to increase, The form is principal mode present in amyloid patch.Make overexpressed disease related mutants form with mankind A β APP transgenic mice it is immune can reduce patch burden and relevant diseases (Schenk etc., 1999, Nature400：173- 177；WO99/27944).For A β antibody periphery apply also reduce brain in patch burden (Bard etc., 2000, Nature Medicine6(8)：916-919；WO2004/032868；WO00/72880).

Antibody therapy provides a kind of promising method for the treatment and prevention of Alzheimer's.However, with bag The human clinical trial for including A β 1-42 vaccine progress suspends due to the meningoencephalitis in patient's subset.Orgogozo etc., Neruology61：7-8(2003)；Ferrer etc., Brain Pathol.14：11-20(2004).Resisted with the anti-A β of N-terminal specificity Body, which carries out passive immunity, causes diffusivity amyloid to substantially reduce, but the micro- bleeding frequency increase of brain of transgenic mice. Pfeifer etc., Science298：1379(2002).There is still a need for the toxicity with reduction be used for treat Alzheimers The improved antibody and/or immune conjugate of disease.

It is prepared for including the DNL for the alemtuzumab for being connected to interferon-λ 1 according to embodiment 9^TMCompound.With 2mg agent Measure to human patientses of the diagnosis with Alzheimer's and intravenously apply interferon-antibody complex, twice a week, continue 4th, 8 or 12 weeks.Effect is measured by neuropsychological test, the test includes the complete neuropsychologicals of ADAS-cog and CERAD Learn test.

In all patients in addition to lowest dose level treatment group, observe that ADAS-cog's is slight after the processing of 12 weeks (15%) improve.Similar result of study is observed for mini-mentalstate examination (MMSE).In 4/10ths patient, Vision structuring capacity is improved.Not it was observed that the serious ill-effect that interferon-antibody is applied.

According to embodiment 9 be prepared for comprising be connected to interferon-λ 1 meter La Zhu monoclonal antibodies (anti-CD74 humanization IgG) or its The DNL of Fab fragments^TMCompound.With the dosage of 2mg twice a week to 75 years old women's vein with early stage Alzheimer's It is interior to apply this compound, continue 8 weeks.Some slight nauseas and low blood pressure signs are noticed after each transfusion, this with Disappear afterwards in 3 days.By Cognitive Aptitude Test (ADAS-cog) and the complete neuropsychological tests of CERAD, in baseline and control After treatment effect is measured at 2 weeks and 8 weeks.As a result indicate, cognitive ability and general psychology and exchange status improve 20%.4 months After repeat the treatment, it has well tolerable property, and patient shows the maintenance that cognitive ability improves, including vision structuring capacity.

It is prepared for according to embodiment 9 comprising the hL243 (anti-HLA-DR humanization IgG) or its Fab for being connected to interferon-λ 1 The DNL of fragment^TMCompound.Intravenously applied to 82 years old women with Alzheimer's with the dosage of 1mg twice a week This compound, continue 8 weeks.Some slight nauseas and low blood pressure sign are noticed after each transfusion, this is in subsequent 3 days Disappear.By Cognitive Aptitude Test (ADAS-cog) and the complete neuropsychological tests of CERAD, in baseline and 2 weeks after treatment Effect is measured during with 8 weeks.As a result indicate, cognitive ability and general psychology and exchange status improve 17%.Being repeated after 4 months should Treatment, it has well tolerable property, and patient shows the maintenance that cognitive ability improves, including vision structuring capacity.

Embodiment 11. is used for (Fab) for treating asthma₂- interferon-λ 1 DNL constructs

Asthma is a kind of heterogeneous familial disease, it is characterised in that high response of the tracheal bronchus for stimulation.Face On bed, asthma show as tracheobronchial narrow, secretion extensively it is thick it is viscous, have difficulty in breathing, cough and pant, so as to lead Airway resistance increase, lung and chest is caused excessively to expand, ventilate and the spatial abnormal feature of lung blood flow.The disease shows as asymptomatic stage Between be interspersed with acute symptom stage of attack.Acute attack causes anoxic, and may be fatal.About the 3% of total world population suffers from The disease.SOA may by triggering antigen presence and level, environmental factor, occupational factor, physical demands and mood Stress and aggravate.Although asthma can use methyl xanthine (such as theophylline), beta-adrenergic activator (such as catecholamine, Resorcinol, bigcatkin willow aglycon and ephedrine), glucocorticoid (such as hydrocortisone), mast cell threshing inhibitor (i.e. color Ketone such as nasmil) and anticholinergic (such as atropine) treatment, but still need the improved method for treating asthma And composition.

There is flu and the acute attack of its asthma in one 80 years old male's Bronchial Asthmas, and frequently cough produces big The thick yellow mucus of amount.Sucked twice daily by aerosol to him through 6 day time and apply (15)-λ 1.Such as institute in embodiment 9 State and prepare antibody-interferon compound.Within several hours of first time suction, his breathing is obviously improved, and cough is infrequently. At the end of this therapeutic process, his physical demands situation seems to be improved, emotional state also in this way, because he Breathing has had improvement and mucus cough substantially reduces.

Embodiment 12. is used for (Fab) for treating multiple sclerosis₂- interferon-λ 1 DNL constructs

Multiple sclerosis (MS) is a kind of autoimmune disease, and wherein autoreactive T cell passes through blood-brain barrier simultaneously Myelin is attacked, inflammation is produced and causes demyelinate and axonal degeneration.The uncertain hair of neurological dysfunction in person between twenty and fifty It is that body and cognitive disorder gradually accumulate in discrete attack (relapsing) or with the time after making.Although also controlled without known Curing method, but interferon-beta therapy is the first-line treatment currently used for recurrent remission form multiple sclerosis, and it is in recurrence rate Effect is shown in terms of reduction.However, IFN therapies are associated with the notable incidence of disease, have such as influenza-like symptom, bone marrow suppression, The side effect such as the development or deterioration of autoimmune disease, Neutrophilic granulocytopenia, decrease of platelet and neuropsychiatric effects.

It is prepared for including the DNL for the alemtuzumab for being connected to interferon-λ 1 as described in example 9 above^TMCompound.Every four weeks 2mg compounds are applied to 64 years old women with Relapsing Multiple Sclerosis disease (RMS).After the treatment of 12 months, compound Observe that annual recurrence rate declines and the risk of disability continuing advances reduces in the case of thing.Compared with baseline measurement, pay attention to Gadolinium enhancing focus to the scanning of each T1 weighted mris substantially reduces.Side effect includes 1/2 grade of infusion reaction, 1 grade of low blood pressure and had A little allergy, these all can well be controlled with corticosteroid.

Claims (27)

1. use of the interferon-antibody complex in the medicine for preparing treating cancer, virus infection, asthma or multiple sclerosis On the way, it includes：

A) interferon-antibody complex is obtained, it is included：

I) the first fusion protein, it includes the interferon-λ of anchoring domain (AD) part for being connected to AKAP protein；

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

B) it is compound using the interferon-antibody to the subject with cancer, virus infection, asthma or multiple sclerosis Thing；

The DDD parts of two of which copy form dimer, and it is described to be formed that the dimer is incorporated into the AD parts Compound, and

Wherein described first fusion protein is SEQ ID NO：The modules of AD2-IFN- λ 1 or SEQ ID NO shown in 96：Shown in 97 AD2-IFN- λ 1-C171S modules, second fusion protein is hRS7, hMN15, hL243, c225, alemtuzumab or rice are drawn The Fab-DDD2 modules of pearl monoclonal antibody.

2. purposes as claimed in claim 1, it further comprises applying other therapeutic agents to the subject.

3. purposes as claimed in claim 2, wherein the therapeutic agent is selected from by secondary antibody, secondary antibody fragment, toxin, swashed Element, cell factor, chemotactic factor (CF), ASON, siRNA, RNAi, radionuclide, boron compound, sensitising agent, anti-blood Pipe generating agent and the group for promoting apoptosis agent composition.

4. purposes as claimed in claim 2, wherein the therapeutic agent is medicine.

5. purposes as claimed in claim 2, wherein the therapeutic agent is immunomodulator.

6. purposes as claimed in claim 2, wherein the therapeutic agent is the second interferon or enzyme.

7. purposes as claimed in claim 4, wherein the medicine is selected from the group consisted of：5 FU 5 fluorouracil, A Pu Halt, azaribine, Anastrozole, anthracycline, bendamustine, bleomycin, bortezomib, Bruton kinase inhibitors, Bryostatin-1, busulfan, calicheamycin, camptothecine, carboplatin, 10-hydroxycamptothecine, BCNU, Celebrex, benzenebutanoic acid nitrogen Mustard, cis-platinum (CDDP), Cox-2 inhibitor, Irinotecan (CPT-11), SN-38, carboplatin, carat Qu Bin, camptothecin, ring phosphorus Acid amides, cytarabine, Dacarbazine, Docetaxel, dactinomycin D, daunorubicin, adriamycin, 2- pyrrolinodoxorubicins (2P-DOX), cyano-morpholine base adriamycin, doxorubicin glucuronide, epirubicin glucuronide, Estramustine, the white poison of table ghost Element, estrogen receptor binding agents, Etoposide (VP16), Etoposide glucosiduronic acid, etoposide phosphate, floxuridine (FUdR), 3 ', 5 '-O- dioleoyls-FudR, fludarabine, Flutamide, farnesyl-protein transferase inhibitor, Ji Xita Shore, hydroxycarbamide, idarubicin, ifosfamide, leunase, lenalidomide, formyl tetrahydrofolic acid, lomustine, Mustargen, melphalan, purinethol, Ismipur, methotrexate (MTX), mitoxantrone, mithramycin, mitomycin, mitotane, promise Wei Ben, nitroso ureas, plicamycin, procarbazine, taxol, Pentostatin, PSI-341, Raloxifene, Semustine, chain Urea rhzomorph, TAM, taxol, Temozolomide, anti-platinum, Thalidomide, thioguanine, thio-tepa, Teniposide, topology are replaced Health, tyrosine kinase inhibitor, uracil mastard, vinorelbine, vincaleukoblastinum, vincristine, vinca alkaloids, LFM-A13, Dasatinib, Imatinib and AMN107.

8. purposes as claimed in claim 3, wherein the toxin is selected from by ricin (WA), abrin, alpha toxin, Saponaria officinalis Element, ribalgilase (RNase), ranpirnase, DNA enzymatic I, staphylococcus enterotoxin-A, pokeweed antiviral protein, white tree poison The group that element, diphtheria toxin, pseudomonas exotoxin and pseudomonas endotoxin form.

9. purposes as claimed in claim 3, wherein the anti-angiogenic agent is selected from the group consisted of：Vascular study Element, bar chalone, canstatin, mammary gland silk suppression albumen, anti-VEGF antibody, anti-P1GF antibody, laminin peptide, fiber connection Albumen, PAI, tissue inhibitor of metalloproteinase, interferon, interleukin 12, IP-10, Gro- β, thrombospondin, 2ME2, proliferin GAP-associated protein GAP, carboxyl acyl aminotriazole, CM101, Ma Li Ma Sita, pentosane polysulfate ester, angiopoietin-2, interferon-' alpha ', Antibiotic TAN 420F, PNU145156E, 16K lactation plain piece Section, linomide (roquinimex), Thalidomide, PTX, genistein, TNP-470, endostatin, taxol, Accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 and minot ring Element.

10. purposes as claimed in claim 1, wherein the amino acid sequence of the DDD parts is selected from the group consisted of： 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.

11. purposes as claimed in claim 1, wherein the interferon-antibody complex than the single interferon, individually The antibody or not conjugated interferon and antibody combination it is more effective.

12. purposes as claimed in claim 1, wherein the cancer is selected from the group consisted of：Acute lymphoblast Property leukaemia, acute myelocytic leukemia, courage cancer, breast cancer, cervix cancer, chronic lymphocytic leukemia, chronic marrow Cell leukemia, colorectal cancer, carcinoma of endometrium, cancer of the esophagus, stomach cancer, head and neck cancer, Hodgkin lymphoma, lung cancer, first shape Gland cephaloma, NHL, Huppert's disease, kidney, oophoroma, cancer of pancreas, glioma, melanoma, Liver cancer, prostate cancer and carcinoma of urinary bladder.

13. purposes as claimed in claim 1, wherein the virus infection is selected from the group consisted of：Human immunity lacks Fall into virus (HIV), herpesviral, herpes simplex virus, vaccinia virus, cytomegalovirus, rabies viruses, influenza virus, rhinopathy Poison, hepatitis type B virus, HCV, sendai virus, feline leukaemia virus, reovirus, poliovirus, The tiny sample virus of human serum, simian virus 40, Respiratory Syncytial Virus(RSV), mouse mammary tumor virus, varicella-zoster disease Poison, dengue virus, rubella virus, measles virus, adenovirus, human T-leukemia virus, epstein-Barr virus, mouse Quasi-leukemia virus, mumps virus, vesicular stomatitis virus, sindbis alphavirus, lymphocytic choriomeningitis disease Poison, verrucosis poison and blue tongue rims.

14. purposes as claimed in claim 13, wherein the virus infection is chronic hepatitis C infection.

15. purposes as claimed in claim 1, wherein the disease is asthma or multiple sclerosis.

16. purposes as claimed in claim 15, wherein the disease is asthma and the compound is to apply to come by nose Apply.

17. purposes as claimed in claim 16, wherein the antibody or antibody fragment are anti-TNF, anti-CD74 or anti-MIF antibody Or antibody fragment.

A kind of 18. composition for including compound, wherein the compound includes：

A) the first fusion protein, it includes the interferon-λ of anchoring domain (AD) part for being connected to AKAP protein；With

B) the second fusion protein, its include be connected to human kinase protein A (PKA) RI, RI β, RII or RII β dimerization and stop By the antibody or antigen binding antibody fragment of domain (DDD) part；And

The DDD parts of two of which copy form dimer, and it is described to be formed that the dimer is incorporated into the AD parts Compound, and

Wherein described first fusion protein is SEQ ID NO：The modules of AD2-IFN- λ 1 or SEQ ID NO shown in 96：Shown in 97 AD2-IFN- λ 1-C171S modules, second fusion protein is hRS7, hMN15, hL243, c225, alemtuzumab or rice are drawn The Fab-DDD2 modules of pearl monoclonal antibody.

19. composition as claimed in claim 18, it further includes the other therapeutic agents selected from the group consisted of： Secondary antibody, secondary antibody fragment, toxin, hormone, cell factor, chemotactic factor (CF), ASON, siRNA, RNAi, put Penetrating property nucleic, boron compound, sensitising agent, anti-angiogenic agent and rush apoptosis agent.

20. composition as claimed in claim 18, it further includes other therapeutic agents, and the other therapeutic agents are medicines.

21. composition as claimed in claim 18, it further includes other therapeutic agents, and the other therapeutic agents are immune tune Save agent.

22. composition as claimed in claim 18, it further includes other therapeutic agents, and the other therapeutic agents are enzymes.

23. composition as claimed in claim 20, wherein the medicine is selected from the group consisted of：5 FU 5 fluorouracil, APL, azaribine, Anastrozole, anthracycline, bendamustine, bleomycin, bortezomib, Bruton kinase inhibitions Agent, Bryostatin-1, busulfan, calicheamycin, camptothecine, carboplatin, 10-hydroxycamptothecine, BCNU, Celebrex, benzene fourth Sour mustargen, cis-platinum (CDDP), Cox-2 inhibitor, Irinotecan (CPT-11), SN-38, carboplatin, carat Qu Bin, camptothecin, Endoxan, cytarabine, Dacarbazine, Docetaxel, dactinomycin D, daunorubicin, adriamycin, 2- pyrrolinyl Ah mould Plain (2P-DOX), cyano-morpholine base adriamycin, doxorubicin glucuronide, epirubicin glucuronide, Estramustine, table ghost are white Toxin, estrogen receptor binding agents, Etoposide (VP16), Etoposide glucosiduronic acid, etoposide phosphate, floxuridine (FUdR), 3 ', 5 '-O- dioleoyls-FudR, fludarabine, Flutamide, farnesyl-protein transferase inhibitor, Ji Xita Shore, hydroxycarbamide, idarubicin, ifosfamide, leunase, lenalidomide, formyl tetrahydrofolic acid, lomustine, Mustargen, melphalan, purinethol, Ismipur, methotrexate (MTX), mitoxantrone, mithramycin, mitomycin, mitotane, promise Wei Ben, nitroso ureas, plicamycin, procarbazine, taxol, Pentostatin, PSI-341, Raloxifene, Semustine, chain Urea rhzomorph, TAM, taxol, Temozolomide, anti-platinum, Thalidomide, thioguanine, thio-tepa, Teniposide, topology are replaced Health, tyrosine kinase inhibitor, uracil mastard, vinorelbine, vincaleukoblastinum, vincristine, vinca alkaloids, LFM-A13, Dasatinib, Imatinib or AMN107.

24. composition as claimed in claim 19, wherein the toxin be selected from by ricin (WA), abrin, alpha toxin, Saporin, ribalgilase (RNase), ranpirnase, DNA enzymatic I, staphylococcus enterotoxin-A, pokeweed antiviral protein, Bai Shu The group that toxin, diphtheria toxin, pseudomonas exotoxin and pseudomonas endotoxin form.

25. composition as claimed in claim 19, wherein the anti-angiogenic agent is selected from the group consisted of：Blood vessel Inhibin, bar chalone, canstatin, mammary gland silk suppression albumen, anti-VEGF antibody, anti-P1GF antibody, laminin peptide, fiber Connect albumen, PAI, tissue inhibitor of metalloproteinase, interferon, interleukin 12, IP- 10th, Gro- β, thrombospondin, 2ME2, proliferin GAP-associated protein GAP, carboxyl acyl aminotriazole, CM101, horse Immediately take charge of he, pentosane polysulfate ester, angiopoietin-2, interferon-' alpha ', Antibiotic TAN 420F, PNU145156E, 16K prolactin(PRL Fragment, linomide (roquinimex), Thalidomide, PTX, genistein, TNP-470, endostatin, taxol, Accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 and minot ring Element.

26. composition as claimed in claim 18, wherein the amino acid sequence of the DDD parts is selected from the group consisted of Group：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.

27. composition as claimed in claim 18, wherein the interferon-antibody complex than the single interferon, The combination of the single antibody or not conjugated interferon and antibody is more effective.