CN105566498A - Bispecific immunocytokine dock-and-lock (dnl) complexes and therapeutic use thereof - Google Patents

Abstract

The present invention concerns methods and compositions for forming cytokine-antibody complexes using dock-and-lock technology. In preferred embodiments, the bispecific immunocytokine DNL construct comprises an IgG antibody attached to a Fab antibody fragment and a cytokine, wherein the IgG and the Fab bind to different target antigens which may be expressed on the same target cell. The bispecific immunocytokine DNL construct exhibits improved pharmacokinetics, with a longer serum half-life and significantly greater efficacy compared to cytokine alone, antibody alone, unconjugated cytokine plus antibody or even other types of cytokine-antibody DNL constructs. In a most preferred embodiment the construct comprises an anti-CD20 IgG antibody conjugated to an anti-HLA-DR Fab and IFNa2b, although other combinations of antibodies, antibody fragments and cytokines may be used to form the subject DNL complexes.

Description

The stop of dual specific immune cell factor-with-lock (DNL) mixture and therapeutic use thereof

The application is the divisional application of following application: the applying date: on August 27th, 2010; Application number: 201080038548.5 (PCT/US2010/046889); Denomination of invention: the same.

cross

This application claims the submit on April 6th, 2010 the 12/754th, No. 740 U.S. Patent applications; Submit on April 5th, 2010 the 12/754th, No. 140 U.S. Patent applications; Submit on April 1st, 2010 the 12/752nd, No. 649 U.S. Patent applications; Submit on March 25th, 2010 the 12/731st, No. 781 U.S. Patent applications; Submit on December 22nd, 2009 the 12/644th, No. 146 U.S. Patent applications; With the submit on August 31st, 2009 the 61/238th, the right of priority of No. 424 U.S. Provisional Patent Application.Each priority application is incorporated herein by reference in their entirety.

government-funded

This working portion is supported by the fund 2R44CA108083-02A2 of the National Cancer Institute from NIH.Federal government can have some right of the present invention.

Sequence table

The application comprise by EFS-Web with ASCII fromat submit to and the sequence table be incorporated herein by reference in their entirety.The described ASCII being created on September 14th, 2010 copies called after IBC128WO.txt and size is 39,795 bytes.

Invention field

The present invention relates to composition and the using method of bi-specific antibody immune cell factor DNL construct, described construct comprises the cytokine of the first and second antibody or antigen binding antibody fragment and one or more copy.More commonly, the present invention relates to composition and the using method of any DNL construct, in described construct, use DDD described below (dimerization and dockerin domain) and AD (anchoring domain) conjugation techniques 3 kinds of different effect thing parts to be linked together.First and second antibody or the different target antigen of its fragment preferred combination two kinds.Cytokine is delivered to target cell, tissue or organ by using of dual specific immune cell factor DNL construct efficiently that comprise the therapeutic cells factor, allows to improve pharmacokinetics, dosage regimen and/or effect simultaneously.The stronger effect for target cell of the cytokine of control antibodies is combined and be conjugated to the display of dual specific immune cell factor construct than the unconjugated of independent parental generation antibody, independent cytokine, antibody and cytokine.In a more preferred embodiment, DNL construct can comprise the interferon-' alpha ' 2b be connected with anti-CD 20 IgG and anti-HLA-DRFab.In the most preferred embodiment, anti-CD 20 IgG be veltuzumab and anti-HLA-DRFab derived from Humanized L 243 antibodies.Described DNL mixture shows the high toxicity to human lymphoma cell, multiple myeloma cells and other hemopoietic cancer (hematopoieticcancer) in vitro and in vivo.But it will be apparent to those skilled in the art that DNL mixture of the present invention can comprise the specific antibody of target antigen or any combination of antibody fragment had for being expressed by any tumour, autoimmune disorder cell or other diseased cells.Similarly, DNL mixture of the present invention can be used for sending any therapeutic cells factor being used for the treatment of and being permitted various diseases such as cancer, immune dysfunction or autoimmune disorder.Those skilled in the art will understand further, and bispecific compound of the present invention is not limited to sending of cytokine, but also can provide the high-efficiency delivery of any therapeutic protein, peptide or other therapeutic effects thing part known in the art.

Background of invention

In the U.S., within 2009, have 65, the new cases of 980 routine non-He Jiejin lymphomas (NHL) and have 19,500 people die from this disease (people such as Jemal, CACancerJClin2009; 59:225-49).The first-line treatment of about half NHL patient is not succeeded and is rarely had (people such as McLaughlin, the JClinOncol1998 of healing; 16:2825-33).Except NHL, also have 20,580 routine multiple myeloma (MM) new cases and have 10,580 people die from this disease (people such as Jemal, CACancerJClin2009; 59:225-49).The clinical activity of interferon-' alpha ' (IFN α) is determined in NHL treatment (people such as Armitage, BoneMarrowTransplant2006; 38:701-2; AnnOncol2000; 11:359-61), and IFN α is added into Rituximab immunotherapy and has shown some clinical favourable aspect (people such as Davis, ClinCancerRes2000; 6:2644-52; The people such as Kimby, LeukLymphoma2008; 49:102-12).Obtainable data show IFN α improve MM patient without progression of disease survival rate, but benefit is very little and it uses because of toxicity still disputable (Gisslinger and Kees, WienKlinWochenschr2003; 115:451-61).

Report the animal model (people such as Ferrantini of interferon-' alpha ' (IFN α) in cancer, 1994, and the human cancer patient (people such as Gutterman JImmunol153:4604-15), 1980, AnnInternMed93:399-406) in all there is Anti-tumor activity.IFN α can produce multiple direct Anti-tumor effect, comprise the downward of oncogene, the enhancing of Immune discrimination that the rise of tumor suppressor gene, the tumor surface MHCI type protein expression through increasing produce, apoptotic enhancing and to the sensitization of the chemotherapeutic (people such as Gutterman, 1994, PNASUSA91:1198-205; The people such as Matarrese, 2002, AmJPathol160:1507-20; The people such as Mecchia, 2000, GeneTher7:167-79; The people such as Sabaawy, 1999, IntJOncol14:1143-51; The people such as Takaoka, 2003, Nature424:516-23).

For some tumour, IFN α has direct and strong anti-multiplication effect (people such as Grimley, 1998Blood91:3017-27) by the activation of STAT1.Indirectly, IFN α can inhibiting angiogenesis (Sidky and Borden, 1987, and stimulation of host immunocyte CancerRes47:5155-61), this is vital for overall antitumor reaction but is greatly underestimated (the people such as Belardelli always, 1996, ImmunolToday17:369-72).IFN α is by medullary cell (people such as Raefsky, 1985, JImmunol135:2507-12; The people such as Luft, 1998, JImmunol161:1947-53), T cell (people such as Carrero, 2006, JExpMed203:933-40; The people such as Pilling, 1999, EurJImmuol29:1041-50) and the effect and having of B cell (people such as Le, 2001, Immunity14:461-70) immunoreactive pleiotropy is affected.As the important conditioning agent of natural immune system, the quick differentiation of IFN α inducing dendritic cell and activation (people such as Belardelli, 2004, CancerRes64:6827-30; The people such as Paquette, 1998, JLeukocbiol64:358-67; The people such as Santini, 2000, and strengthen the cytotoxicity of NK cell, migration, cytokine production and antibody dependent cellular cytotoxicity (ADCC) (people such as Biron, 1999, AnnuRevImmunol17:189-220 JExpmed191:1777-88); The people such as Brunda 1984, CancerRes44:597-601).

IFN α is hindered because of its of short duration circulating half-life and systemic toxicity as the hope of cancer therapeutic agent is all the time main.The cycling time of the PEGization form display increase of IFN α 2, which increase their biological effect (Harris and Chess, 2003, NatRevDrugDiscov2:214-21; The people such as Osborn, 2002, JPharmacolExpTher303:540-8).IFN α can provide the benefit similar to PEGization to the fusion of monoclonal antibody (MAb), the circulating half-life comprising the renal clearance of minimizing, the solubleness of raising and stability and significantly increase.The direct clinical benefit of this fusion needs lower frequency and lower dosage, thus allow the treatment concentration of prolongation.

Use and IFN α target tumor significantly can be increased its tumor adhesion (accretion) for the MAb of tumor associated antigen (TAA) and store (retention) with its systemic concentration of limit, thus increase therapeutic index.The tumor levels of the increase of IFN α can increase its direct antiproliferative, apoptosis and anti-angiogenic activity, and causes and concentrated anti tumor immune response.In fact, the research of the mouse of homogenic type mouse IFN alpha-secretase transgenic tumor is used to show immune response people such as (, 2007, Biochimie89:884-93) Ferrantini of the enhancing caused by the partial concn of IFN α.

CD20 is the attracting candidate TAA of the treatment for the B cell lymphoma using MAb-IFN α to carry out.Utilize the anti-CD 20 immunotherapy of Rituximab to be one of most successful treatment of lymphoma, it has relatively low toxicity (people such as McLaughlin, 1998, JClinOncol16:2825-33).Because Rituximab is the chimeric antibody (people such as Cheson that can show immunogenicity in some PATIENT POPULATION and initial application be had to quite long Infusion Time, 2008, NEJM359:613-26), so the better material standed for for CD20-target is humanization MAb, veltuzumab (the people such as Stein, 2004, ClinCancerRes10:2868-78).

The combination therapy being in use Rituximab under clinical evaluation and IFN α has at present shown effect (people such as Kimby, 2008, LeukLymphoma49:102-12 of the raising higher than independent Rituximab; The people such as Salles, 2008, Blood112:4824-31).Some favourable aspect of these these combinations of research display and the shortcoming relevant to IFN α.Except utilizing the infusion of Rituximab once in a week, patient also will use weekly IFN α 3 times usually in time several months, suffers influenza-like symptom, and described symptom is the common adverse effect relevant to IFN α therapy and limits tolerable dose.Antibody-IFN alpha conjugate can allow to use single agents with less dosage with lower frequency, limits or eliminates side effect, and can produce much higher effect.But lymphoma and the leukemia expection of expressing or do not express CD20 hardly have resistance to the treatment of the anti-CD 20-IFN α construct utilizing immunoconjugates.Exist in this area by IFN-α or other two or more different tumor associated antigen of therapeutic cells factor target such as CD20 and HLA-DR, the needs of the dual specific immune cell factor construct of the treatment of more effective anti-many kinds of hematopoietic malignancies and other malignant tumour being provided.

Human leucocyte antigen (HLA)-DR (HLA-DR) is one of 3 kinds of isotypes of major histocompatibility complex (MHC) II type antigen.HLA-DR is highly expression on multiple hematopoietic malignancies and some solid tumor, and by actively for lymphoma treating (people such as Brown, 2001, ClinLymphoma2:188-90 based on antibody; The people such as DeNardo, 2005, ClinCancerRes11:7075s-9s; The people such as Stein, 2006, Bloood108:2736-44).Preliminary study shows anti-HLA-DRmAb more effective force more obvious than other naked mAb with Present clinical benefit people such as (, unpub result) Stein in the in vitro and in vivo experiment of lymphoma, leukemia and multiple myeloma.HLA-DR also above to express people such as (, 2003, SeminOncol30:465-75) Dechant at one group of normal immunocyte (comprising the T cell of B cell, monocyte/macrophage, youth Ge Erhansi cell, dendritic cell and activation).

summary of the invention

The present invention relates to composition and the using method of stop-(DNL) construct (mixture) that locks comprising three kinds or more kind different effect thing part such as antigen, antibody fragment and cytokine.But, those skilled in the art will know that DNL construct is not limited to this and the effector part used can comprise any protein, peptide or other molecule that can be connected to DDD or AD part.Protein, peptide, antibody, antibody fragment, immunomodulator, cytokine, hormone, enzyme, antisense oligonucleotide such as siRNA, toxin such as rnase, heterologous antigen, polyoxyethylene glycol (PEG) and other polymkeric substance, anti-angiogenic agent, cytotoxic agent, short apoptosis agent and other known therapeutical agent is included but not limited to for the effector part in DNL construct.

Preferred embodiment relates to the DNL construct of the cytokine comprising three kinds of different effect thing part-the first and second antibody or antibody fragment and one or more copy.In the most preferred embodiment, DNL construct comprises anti-CD 20 antibodies, such as veltuzumab, anti-HLA-DR antibody fragment such as hL243 and cytokine such as IFN-α 2b.This type of DNL construct is highly effective for the treatment of hematopoietic malignancies and expression CD20, HLA-DR or both other tumours.Although each component of multi-functional mixture (veltuzumab, anti-HLA-DRFab, with IFN-α 2b) there is Anti-tumor activity independently, but the display of the construct of described combination is than any single component or the larger effect of the mixture of single component used with unconjugated form.

In particular embodiments, DNL construct can comprise the anti-HLA-DR antibody of humanization or its fragment, such as hL243 antibody, it comprises the heavy CDR sequences CDR1 (NYGMN being connected to people's Antibody framework (FR) and constant-region sequences, SEQIDNO:1), CDR2 (WINTYTREPTYADDFKG, and CDR3 (DITAVVPTGFDY SEQIDNO:2), and CDR sequence CDR1 (RASENIYSNLA SEQIDNO:3), SEQIDNO:4), CDR2 (AASNLAD, and CDR3 (QHFWTTPWA SEQIDNO:5), SEQIDNO:6) (see people, such as, 7th, 612, No. 180 United States Patent (USP)s, embodiment part is incorporated to herein by reference).In preferred embodiments, Humanized L 243 antibodies also can comprise one or more residues of the one or more residue from the Framework residues 27,38,46,68 and 91 substituted of mouse L243 (mL243) heavy chain and/or the Framework residues 37,39,48 and 49 substituted from mL243 light chain.Can in American type culture collection, Rockville, MD (see accession number ATCCHB55) obtain mL243.

In other specific embodiment, DNL construct can comprise humanization anti-CD 20 antibodies or its fragment, such as veltuzumab, and it comprises variable region of light chain CDR1 (RASSSVSYIH, SEQIDNO:7); CDR2 (ATSNLAS, SEQIDNO:8); With CDR3 (QQWTSNPPT, SEQIDNO:9); And variable region of heavy chain CDR1 (SYNMH, SEQIDNO:10); CDR2 (AIYPGNGDTSYNQKFKG, and CDR3 (STYYGGDWYFDV (SEQIDNO:95) or VVYYSNSYWYFDV SEQIDNO:11), SEQIDNO:12) (see, such as, 7th, 435, No. 803 United States Patent (USP)s, embodiment part is incorporated to herein by reference).

In a more particular embodiment, DNL construct can comprise humanIFN-α 2b aminoacid sequence.The clone comprising this type of sequence can be commercially available from multiple source, and such as total length people IFN α 2bcDNA clones (UltimateORF people clones cat#HORF01CloneIDIOH35221, Invitrogen, Carlsbad, CA).

In various embodiments, DNL construct can comprise one or more antibody combining the antigen except CD20 and/or HLA-DR or its fragments.In preferred embodiments, described antigen can be selected from carbonic anhydrase IX, CCCL19, CCCL21, CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, AFP, PSMA, CEACAM5, CEACAM-6, B7, the ED-B of fibronectin, factor H, FHL-1, Flt-3, folacin receptor, GROB, HMGB-1, hypoxia inducible factor (HIF), HM1.24, insulin-like growth factor-i (ILGF-1), IFN-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-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5, PAM4 antigen, NCA-95, NCA-90, Ia, HM1.24, EGP-1, EGP-2, HLA-DR, tenascin, Le (y), RANTES, T101, TAC, Tn antigen, Thomson-Friedenreich antigen, tumor necrosis antigens, TNF-α, TRAIL acceptor (R1 and R2), VEGFR, EGFR, PlGF, complement factor C_3, C3a, C3b, C5a, C5 and oncoprotein.

Available exemplary antibodies includes but not limited to hR1 (anti-IGF-1R, submit on December 3rd, 2010 the 12/722nd, No. 645 U.S. Patent applications), hPAM4 (anti-Saliva Orthana, 7th, 282, No. 567 United States Patent (USP)s), hA20 (anti-CD 20, 7th, 251, No. 164 United States Patent (USP)s), hA19 (anti-CD19, 7th, 109, No. 304 United States Patent (USP)s), hIMMU31 (anti-AFP, 7th, 300, No. 655 United States Patent (USP)s), hLL1 (anti-CD74, 7th, 312, No. 318 United States Patent (USP)s), hLL2 (anti-CD22, 7th, 074, No. 403 United States Patent (USP)s), hMu-9 (anti-CSAp, 7th, 387, No. 773 United States Patent (USP)s), hL243 (anti-HLA-DR, 7th, 612, No. 180 United States Patent (USP)s), hMN-14 (anti-CEACAM5, 6th, 676, No. 924 United States Patent (USP)s), hMN-15 (anti-CEACAM6, 7th, 541, No. 440 United States Patent (USP)s), hRS7 (anti-EGP-1, 7th, 238, No. 785 United States Patent (USP)s) and hMN-3 (anti-CEACAM6, 7th, 541, No. 440 U.S. Patent applications), the embodiment part of each patent quoted or application is incorporated to herein by reference.It will be apparent to those skilled in the art that this list is not restrictive and can uses any known antibody, as discussed in more detail below.

The Examples of cytokines that can be integrated into DNL construct includes but not limited to MIF (macrophage migration inhibiting factor), HMGB-1 (high mobility group frame albumen 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-23, IL-24, CCL19, CCL21, IL-8, MCP-1, RANTES, MIP-1A, MIP-1B, ENA-78, MCP-1, IP-10, Gro-β, eotaxin (Eotaxin), interferon-' alpha ',-β,-λ, G-CSF, GM-CSF, SCF, PDGF, MSF, Flt-3 part, erythropoietin, thrombopoietin, CNTF, leptin, oncostatin M, VEGF, EGF, FGF, PlGF, Regular Insulin, hGH, thyrocalcitonin, Factor IX, IGF, somatostatin, tissue plasminogen activator and LIF.The sequence of the person form of each cytokine quoted in this area, be known (see such as ncbi database) and the clone of many Examples of cytokines of encoding can be commercially available from Invitrogen, American type culture collection and other source known in the art.

Multiple embodiments can relate to the purposes that DNL construct of the present invention is used for the treatment of or diagnoses the illness, described disease includes but not limited to non-He Jiejin lymphomas, B cell acute and chronic lymphatic leukemia, Burkitt lymphoma, He Jiejin lymphomas, hairy cell leukemia, acute and chronic myeloid leukemia, t cell lymphoma and leukemia, multiple myeloma, neurospongioma, Walden Si Telun macroglobulinemia, cancer, melanoma, sarcoma, neurospongioma and skin carcinoma.Cancer can be selected from oral carcinoma, gastrointestinal cancer, lung road cancer, lung cancer, mammary cancer, ovarian cancer, prostate cancer, uterus carcinoma, carcinoma of endometrium, cervical cancer, bladder cancer, carcinoma of the pancreas, osteocarcinoma, liver cancer, carcinoma of gallbladder, kidney, skin carcinoma and carcinoma of testis.In addition, DNL construct of the present invention can be used for treatment autoimmune disorder such as acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea (Sydenham'schorea), myasthenia gravis, systemic lupus erythematous, systemic lupus erythematosus, rheumatic fever, polyglandular syndrome, bullous pemphigoid, diabetes, purpura,Henoch-Schonlein (Henoch-Schonleinpurpura), ephritis (post-streptococcalnephritis) after streptococcal infection, erythema nodosum, Gao An (family name) arteritis (Takayasu'sarteritis), Addison disease (Addison'sdisease), rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome (Goodpasture'ssyndrome), thromboangiitis obliterans (thromboangitisubiteran), xerodermosteosis, primary biliary cirrhosis, struma lymphomatosa (Hashimoto'sthyroiditis), thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris, Wei Genashi granuloma (Wegener'sgranulomatisis), membranous nephropathy, amyotrophic lateral sclerosis, myelophthisis, giant cell arteritis/polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriatic or fibrosing alveolitis.In certain embodiments, Subject antibodies can be used for treatment leukemia such as chronic lymphocytic leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia or acute myeloid leukemia.

In one embodiment, pharmaceutical composition of the present invention can be used for treating the experimenter suffering from metabolic trouble such as amyloidosis or nerve degenerative diseases such as alzheimer's disease.In addition, pharmaceutical composition of the present invention can be used for treating the experimenter suffering from immune disorder obstacle.

accompanying drawing is sketched

Following accompanying drawing forms the part of this specification sheets and is used to further illustrate certain embodiments of the present invention.One or more by reference to these accompanying drawings be combined with the detailed description of the specific embodiments to provide herein, can understand described embodiment better.

Fig. 1. the external IFN alpha active of the showed cell factor-MAbDNL construct compared with PEGization or natural IFN α.The specific activity (IU/pmol) of measurement as be shown in the examples.The activity of each trial target of concentration known is inferred from rhIFN α 2b typical curve.At 20-2b (●), the 734-2b (■) of increasing concen-trations, v-mab (zero), v-mab+734-2b (), PEGASYS , PEG-Intron (▲) or 1R-2b deposit grown culture in case, and utilize MTS to measure relative viable cell density.Signal % available from untreated cell is mapped to the logarithm of volumetric molar concentration.Prism software is used to produce dose-response curve and EC ₅₀value.Error bar, SD.(A) based on the reporter-gene assays of cell.(B) the virus protection of EMC virus and A549 cell is utilized to measure.(C) the external lymphoma proliferation assay of Daudi cell is used.(D) the external lymphoma proliferation assay of Jeko-1 cell is used.

Fig. 2. the result of the pharmacokinetic analysis of display Swiss-Webster mouse.Use 20-2b, α 2b-413 to mouse, wear happy can (PEGINTRON) or PEG-IFN alpha-2a (PEGASYS), and in 96 hours in utilize the IFN α 2b concentration of elisa assay serum sample.Curve eliminated by display serum.Serum half-life (T _1/2) supersession rate and average retention time (MRT) be summarized in the table of insertion.

Fig. 3. (A) illustrates the ADCC effector function of 20-2b.Lysis quantitative before, deposit in case at the PBMC of fresh separated, utilize 20-2b, the 22-2b of 5 μ g/ml, v-mab, epratuzumab (e-mab) or h734 incubation Daudi or Raji cell 4 hours.(B) the CDC effector function of 20-2B is shown.Deposit in case at people's complement, utilize 20-2b (●), 734-2b (■) or v-mab (zero) the incubation Daudi cell of serial dilution.Complement control % (number of the viable cell of the test sample compared with only utilizing the cell of complement process) is mapped to the logarithm of nM concentration.Error bar, SD.

Fig. 4. display 20-2b strengthens the consumption from the NHL cell of whole blood.Fresh heparinized human blood is mixed with Daudi or Ramos and utilizes 0.01,0.1 or 20-2b (●), the v-mab (zero) of 1nM, 734-2b (■) or v-mab+734-2b () incubation 2 days.Use the shown effect processed lymphoma and peripheral blood lymphocyte of flow cytometry assessment.Error bar, SD.

Fig. 5. (A) illustrates the therapeutic efficiency of display 20-2b in dissemination Burkitt lymphoma (disseminatedBurkitt ' slymphoma) (Daudi) heteroplastic transplantation model.Gave in female C.B.17SCID mouse vein at the 0th day and use Daudi cell.Process is by 20-2b (●), the 734-2b (■) that provide with single SC dosage, v-mab (zero), PEGASYS or salt solution (X) composition.The number of days arrow of process indicates.Use Prism software analysis survivorship curve.In early days in Daudi model.The single dose using 0.7pmol (solid line) or 0.07pmol (dotted line) to the group of 10 mouse at the 1st day.(B) research carried out in Daudi model to the similar of Fig. 6 (A) but late of display.The single dose using 0.7pmol (solid line), 7pmol (dotted line) or 70pmol (grey lines) to the group of 10 mouse at the 7th day.

Fig. 6. (A) shows the survivorship curve of the therapeutic efficiency of 20-2b in dissemination Burkitt lymphoma (Raji and NAMALWA) heteroplastic transplantation model.Gave in female C.B.17SCID mouse vein at the 0th day and use NHL cell.Process and be made up of 20-2b (●), the 734-2b (■) that give with subcutaneous dosage, v-mab (zero) or salt solution (X).The number of days arrow of process indicates.Use Prism software analysis survivorship curve.Late in Raji model.The group of 10 mouse accepted the dosage of 250pmol at the 5th, 7,9,12,14 and 16 day.(B) research of display to the similar of Fig. 6 (A) but in early days in NAMALWA model.The group of 6 mouse accepted 20-2b or 734-2b of 250pmol dosage or accepted the v-mab of 3.5nmol dosage at the 1st, 5,9,13,17,21 and 25 day at the 1st, 3,5,8,10 and 127 day.

Fig. 7. display uses the result of the mensuration based on cell of the EPO activity of EPO standard, 734-EPO or the EPO-DDD2 process TF1 cell of 72 hours.GraphPadPrism software is used to produce dose response curve and EC ₅₀value.

The biological activity of Fig. 8 .20-C2-2b.A and B, display MAb and MAb-IFN α is to the indirect immunofluorescence of the combination of NHL cell (Raji or RL) that lives.Before the goat anti-human Fc that puts together with PE-detects, deposit in case in 4 DEG C of Incubate cells 1 hour at the shown construct of 5nM (A) or 0.2-50nM (B).MFI, average fluorescent strength; Error bar, 95%CI.(C) IFN α 2 specific activity using the reporter gene assay based on cell to measure is shown as the entire molecule of IU/pmol and the IFN α 2b of IU/pmol.

The apoptosis of Fig. 9 .NHL and MM cell.Utilizing the pre-treatment cell 48 hours of the quantitative annexin of fluidic cell number-V-positive cell %.(A) be 10pM for Daudi:v-mab and hL243 γ 4p; 20-C2-2b, 20-2b-2b and V+L243+2b (mixture of v-mab, hL243 γ 4p and 734-2b-2b) are 1pM.For Jeko-1, all process is carried out on 0.5nM.(B) with 1,0.1 and 0.01nM process CAG.(C) with 20 and 2nM process KMS12-BM.The mixture of V+L243, v-mab and hL243 γ 4p; The mixture of L243+2b, hL243 γ 4p and 734-2b-2b.

Figure 10. the sign of multiple myeloma cell line.(A) antigen density of HLA-DR and CD20 that the myeloma cell selected by fastens.Utilizing hL243 γ 4p, v-mab or hMN-14 (isotype controls MAb) incubation after 30 minutes, utilizing PE-goat anti-human IgG (Fab) to detect cell, utilizing flow cytometry cell.(B) melanoma cell series is to the relative sensitivity of IFN α 2.Before utilizing MTS quantifying live cells, Incubate cells 4 days in 3nM peg-interferon α-2b presence or absence situation.

Figure 11. the vitro cytotoxicity of multiple myeloma.Shown in increasing concen-trations, construct or combination are deposited and are cultivated shown clone in case, utilize MTS to measure relative viable cell density.Signal % available from untreated cell is mapped to the logarithm of volumetric molar concentration.Prism software is used to produce dose-response curve and EC ₅₀value.Error bar, SD.

Figure 12. the consumption of the NHL cell from whole blood of enhancing.Fresh heparinized human blood is mixed with Daudi, incubation 2 days together with α or MAb of Mab-IFN shown in 1nM.Flow cytometry is utilized to assess Daudi, B cell, T cell and monocytic effect.Error bar, SD.

Figure 13. diagram MAb-IFN α is to the vitro cytotoxicity of NHL and MM cell.

detailed Description Of The Invention

Definition

Unless pointed out clearly in addition, otherwise " one " or " one " can refer to " one or more ".

Term as used herein " with " can be used for conjunction (conjunctive) or extract (disjunctive) with "or".That is, unless otherwise stated, these two terms should be understood to be equal to "and/or".

" therapeutical agent " can be used for the atom of disease therapy, molecule or compound.The example of therapeutical agent comprises antibody, antibody fragment, peptide, medicine, toxin, enzyme, nuclease, hormone, immunomodulator, antisense oligonucleotide, siRNA (siRNA), sequestrant, boron compound, photosensitizers, dyestuff and radio isotope.

" diagnostic reagent " is the atom, molecule or the compound that can be used for diagnosing the illness.Useful diagnostic reagent includes but not limited to radio isotope, dyestuff (such as with biotin-streptavidin mixture), contrast medium, fluorescent chemicals or molecule and the toughener (such as paramagnetic ion) for nuclear magnetic resonance (MRI).

" antibody " used herein refers to immunocompetence (i.e. specific binding) part of total length (namely naturally occurring or formed through standard immunoassay globulin gene fragment recombinatorial processes) immunoglobulin molecules (such as IgG antibody) or immunoglobulin molecules, as antibody fragment." antibody " comprises monoclonal antibody, polyclonal antibody, bi-specific antibody, multi-specificity antibody, murine antibody, chimeric antibody, humanized antibody and people's antibody.

" naked antibody " is the antibody or its Fab that are not connected with therapeutical agent or diagnostic reagent.The Fc part of complete naked antibody can provide effector function, such as complement combination and ADCC (see, such as, Markrides, PharmacolRev50:59-87,1998).Naked antibody nationality can comprise apoptosis with other mechanism of inducing cell death.(Vaswani and Hamilton, AnnAllergyAsthmaImmunol81:105-119,1998.).

" antibody fragment " is the part of complete antibody, such as F (ab') ₂, Fab', Fab, Fv, sFv, scFv etc.Regardless of its structure, the antigen that the antigen that antibody fragment combines and complete antibody identify is identical.Antibody fragment comprises fragment (" Fv " fragment be such as made up of heavy chain and variable region of light chain) or the recombinant single chain peptide molecule (wherein light chain is connected (" scFv albumen ") by peptide linker with variable region of heavy chain) of the separation be made up of variable region." single-chain antibody " (being usually abbreviated as " scFv ") interacts by comprising with the V forming antigen-binding site _hand V _lthe polypeptide chain composition of structural domain.V _hand V _lstructural domain is connected by the peptide of 1 to 25 amino-acid residue usually.Antibody fragment also comprises double-chain antibody, three chain antibodies and single domain antibody (dAb).

If when the dosage used has physiological meaning, just can be described as with " treatment significant quantity " administration of antibodies or immunoconjugates preparation or composition as herein described.If the existence of reagent causes the detectable change of the physiological status of recipient subjects, then this reagent just has physiological meaning.In particular embodiments, if when the existence of antibody preparation causes antitumor reaction or alleviates the S&S of autoimmune disorder state, this antibody preparation just has physiological meaning.The effect of physiological significance also can be bring out body fluid and/or the cell immune response of recipient subjects, causes growth-inhibiting or the death of target cell.

Stop and (DNL) method that locks

The specific protein-protein interaction that occurs between adjustment (R) subunit of the protein kinase (PKA) that " stop-and-lock " (DNL) method utilizes cAMP to rely on and the anchoring domain (AD) of A-kinase anchoring protein (AKAP) (people such as Baillie, FEBSLetters.2005; 579:3264.Wong and Scott, Nat.Rev.Mol.CellBiol.2004; 5:959).PKA (playing Main Function in by one of signal transduction path triggered with the combination of R subunit by second messenger cAMP of most scrutiny) is separated (people such as Walsh, J.Biol.Chem.1968 from rabbit first in nineteen sixty-eight; 243:3763).The structure of holoenzyme is made up of two catalytic subunits, and these two subunits maintain inactive form (Taylor, J.Biol.Chem.1989 by R subunit; 264:8443).Find that the isozyme class of PKA has two class R subunits (RI and RII), and each class have α and β isotype (Scott, Pharmacol.Ther.1991; 50:123).R subunit is only separated with stable dimer, and has shown dimerization domain and form (people such as Newlon, Nat.Struct.Biol.1999 by front 44 amino-terminal residue; 6:222).The combination of cAMP and R subunit causes the release of the active catalytic subunit for broad spectrum serine/threonine kinase activity, the described PKA of being released through is directed to selected substrate (people such as Scott, J.Biol.Chem.1990 via the compartmentation of the stop of itself and AKAP; 265; 21561).

Due to an AKAP, microtubule associated protein 2, was characterized (people such as Lohmann, Proc.Natl.Acad.SciUSA.1984 in 1984; 81:6723), the AKAP (Wong and Scott, the Nat.Rev.Mol.CellBiol.2004 that are positioned to different subcellular location (comprising plasma membrane, actin cytoskeleton, nucleus, plastosome and endoplasmic reticulum) more than 50 kinds with different structure has been identified in the species scope of yeast to the mankind; 5:959).The AD of the AKAP of PKA is amphipathic helix (people such as Carr, the J.Biol.Chem.1991 of 14-18 residue; 266:14188).The aminoacid sequence of AD is quite changeable in individual AKAP, and what report is 2 to 90nM (people such as Alto, Proc.Natl.Acad.Sci.USA.2003 to the dimeric binding affinity scope of RII; 100:4445).Enjoyably, AKAP will only in conjunction with dimerization R subunit.People RII, AD are combined to the hydrophobic surface (Colledge and Scott, the TrendsCellBiol.1999 that are formed by 23 n terminal residues; 6:216).Therefore, the dimerization domain of people RII α and AKAP binding domains are all arranged in identical N-terminal 44 aminoacid sequences (people such as Newlon, Nat.Struct.Biol.1999; 6:222; The people such as Newlon, EMBOJ.2001; 20:1651), described aminoacid sequence is referred to herein as DDD.

As the AD of DDD and AKAP of the people RII α of link block (LinkerModule)

We have developed such platform technology, it utilizes the AD of DDD and the AKAP albumen of people RII α to stop in non-covalent complex for by any combination of the entity hereinafter referred to as A and B as a pair excellent link block, non-covalent complex by cysteine residues is introduced DDD and AD strategic location on lock further in the stable structure fettered to promote the formation of disulfide linkage.The general method of " stop-and-lock " method is as follows.By DDD sequence to be connected the entity A that (result produces the first component hereinafter referred to as a) builds with the precursor of A.Because DDD sequence can cause dimeric spontaneous formation, therefore A can by a ₂composition.Entity B is built by AD sequence to be connected (result produces the second component hereinafter referred to as b) with the precursor of B.A ₂in the dimerization primitive of DDD that comprises can produce the docking site that the AD sequence for comprising in b is combined, thus promote a ₂with the easy association of b to be formed by a ₂the trimerization mixture of b composition.Such binding events irreversibly carries out because of subsequent reactions, described subsequent reactions protects described two entities by disulphide bridges covalency, described reaction effectively occurs based on the principle of effective localized concentrations, because initial binding interactions should make to be placed in reactive thiol group on DDD and AD near (people such as Chmura, Proc.Natl.Acad.Sci.USA.2001; 98:8480) to carry out locus specificity connection.Although in certain embodiments, a ₂subunit can comprise two identical effector parts, but in the preferred embodiment described hereinafter, a ₂subunit can comprise different effector parts, and each effector connects with identical DDD sequence.Therefore, trimerization a ₂b mixture can comprise three kinds of different effector parts.

In preferred embodiments, immune cell factor DNL construct can based on a ₂the change of b structure, wherein the first and second effector and DDD part are connected and the 3rd effector and AD are partly connected.Each AD part can in conjunction with two the DDD parts existed with dimeric forms.By connecting DDD and AD away from the functional group of precursor, this type of locus specificity connects also expection and keeps the original activity of precursor.The method is module in essence and can be used for locus specificity potentially and be covalently connected many kinds of substance perhaps.DNL method is disclosed in the 7th, 550, No. 143; 7th, 521, No. 056; 76th, 534, No. 866; In 7th, 527, No. 787 and the 7th, 666, No. 400 United States Patent (USP)s, the embodiment part of each patent is incorporated to herein by reference.

In preferred embodiments, effector part to be connected with DDD or AD part and the protein or peptide, more preferably antibody, antibody fragment or the cytokine that form fusion rotein or peptide.Multiple method for the preparation of fusion rotein is known, comprises nucleic acid synthesis, hybridization and/or amplification to produce the synthesis double-strandednucleic acid of coding object fusion rotein.Usable criterion Protocols in Molecular Biology (see, the people such as such as Sambrook, MolecularCloning, Alaboratorymanual, the 2nd edition, 1989) this type of double-strandednucleic acid is inserted the expression vector being used for fusion rotein and producing.In this type of preferred embodiment, the N-terminal of AD and/or DDD part and effect protein or peptide or C-terminal can be connected.But, those skilled in the art will know that AD or DDD partly can be depending on the chemical property of effector part to the site of the connection of effector part and change, and the part of effector part involves its physiologically active.In the most preferred embodiment, AD or DDD part can occur to the connection of antibody or antibody fragment on the C-terminal of heavy chain subunit, opposing end portions from the molecule of antigen-binding site.But, as hereinafter discuss, the N-terminal to antibody or antibody fragment also can be used to connect, keep antigen-binding activity simultaneously.Technology known in the art can be used such as to use divalence cross-linking reagent and/or other chemically conjugated technology to connect to the locus specificity carrying out multiple effector part.

DDD and AD sequence variants

In certain embodiments, AD and the DDD sequence being integrated into immune cell factor DNL mixture comprises the aminoacid sequence of DDD1 and AD1 hereinafter.In a more preferred embodiment, AD and DDD sequence comprises the aminoacid sequence of DDD2 and AD2, and described sequence promotes the formation of disulfide linkage between DDD and AD part through design.

DDD1

SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQIDNO:13)

DDD2

CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQIDNO:14)

AD1

QIEYLAKQIVDNAIQQA(SEQIDNO:15)

AD2

CGQIEYLAKQIVDNAIQQAGC(SEQIDNO:16)

It will be understood by those skilled in the art that DDD1 and DDD2 comprises the DDD sequence of the people RII alpha-form of protein kinase A.But in selectable embodiment, DDD and AD part can based on the DDD sequence of the people RII alpha-form of protein kinase A and corresponding AKAP sequence, as illustrational in institute in DDD3, DDD3C and AD3 hereinafter.

DDD3

SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK(SEQIDNO:17)

DDD3C

MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK(SEQIDNO：18)

AD3

CGFEELAWKIAKMIWSDVFQQGC(SEQIDNO：19)

Also can design and utilize based on known people RI β and RII beta amino acids sequence other selectable DDD part (see, such as, NCBI accession number NP_001158233 and NP_002727, sequence hereinafter).

People PKARI beta amino acids sequence

MASPPACPSEEDESLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEENRQILARQKSNSQSDSHDEEVSPTPPNPVVKARRRRGGVSAEVYTEEDAVSYVRKVIPKDYKTMTALAKAISKNVLFAHLDDNERSDIFDAMFPVTHIAGETVIQQGNEGDNFYVVDQGEVDVYVNGEWVTNISEGGSFGELALIYGTPRAATVKAKTDLKLWGIDRDSYRRILMGSTLRKRKMYEEFLSKVSILESLEKWERLTVADALEPVQFEDGEKIVVQGEPGDDFYIITEGTASVLQRRSPNEEYVEVGRLGPSDYFGEIALLLNRPRAATVVARGPLKCVKLDRPRFERVLGPCSEILKRNIQRYNSFISLTV(SEQIDNO：20)

People PKARI beta amino acids sequence

MSIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENERKGTARFGHEGRTWGDLGAAAGGGTPSKGVNFAEEPMQSDSEDGEEEEAAPADAGAFNAPVINRFTRRASVCAEAYNPDEEEDDAESRIIHPKTDDQRNRLQEACKDILLFKNLDPEQMSQVLDAMFEKLVKDGEHVIDQGDDGDNFYVIDRGTFDIYVKCDGVGRCVGNYDNRGSFGELALMYNTPRAATITATSPGALWGLDRVTFRRIIVKNNAKKRKMYESFIESLPFLKSLEFSERLKVVDVIGTKVYNDGEQIIAQGDSADSFFIVESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALFGTNMDIVEPTA(SEQIDNO：21)

In other selectable embodiment, the different sequence variants of AD and/or DDD part can be used for the structure of dual specific immune cell factor DNL mixture.The structure-function relationship of AD and DDD structural domain is Investigational theme always.(see, such as, the people such as Burns-Hamuro, 2005, ProteinSci14:2982-92; The people such as Carr, 2001, JBiolChem276:17332-38; The people such as Alto, 2003, ProcNatlAcadSciUSA100:4445-50; The people such as Hundsrucker, 2006, BiochemJ396:297-306; The people such as Stokka, 2006, BiochemJ400:493-99; The people such as Gold, 2006, MolCell24:383-95; The people such as Kinderman, 2006, MolCell24:397-408, the full copy of each document is incorporated to herein by reference).

Such as, the people such as Kinderman (2006) examine the crystalline structure of AD-DDD binding interactions and show that people DDD sequence comprises many conservative amino acid residues (described amino-acid residue is particularly important in dimer is formed and AKAP combines), underline in following sequence.(see people such as Kinderman, Fig. 1 of 2006, described document is incorporated to herein by reference).It will be appreciated by those skilled in the art that in the sequence variants of design DDD sequence, desirably avoid changing any underlined residue, but can substitute carrying out conserved amino acid for dimerization and the not too critical residue of AKAP combination.Therefore, be shown in one sequence for the potential selectable DDD sequence building DNL mixture, wherein " X " represents that conserved amino acid substitutes.Conserved amino acid sequence substitutes and is discussed in greater detail hereinafter, but can comprise such as asparagicacid residue and substitute glutaminic acid residue or leucine or valine residue and substitute isoleucine residues etc.This type of conserved amino acid substitutes and is well known in the art.

From the people DDD sequence of protein kinase A

SH IQ IPPG LTE LLQG YT VE VLRQQPPD LVE FA VE YFTR LREARA(SEQIDNO:13)

XX IX IXXX LXX LLXX YX VX VLXXXXXX LVX FX VX YFXX LXXXXX(SEQIDNO:22)

The people such as Alto (2003) have carried out the bioinformatic analysis of the AD sequence of multiple AKAP albumen to design the RII selectivity AD sequence being called AKAP-IS hereinafter shown, and it has the binding constant to DDD of 0.4nM.AKAP-IS sequence is designed to the peptide antagonists of the AKAP in conjunction with PKA.Wherein the residue substituted in the AKAP-IS sequence of the combination tending to minimizing and DDD underlines in following sequence.Therefore, it will be understood by those skilled in the art that the variant of the function that can play DNL construct is indicated by one sequence, wherein " X " is that conserved amino acid substitutes.

AKAP-IS sequence

QIEYL AKQ IVDN AIQQA(SEQIDNO:15)

XXXXX AXX IVXX AIXXX(SEQIDNO:23)

Similarly, Gold (2006) utilizes crystallography and peptide screening to develop the SuperAKAP-IS sequence hereinafter shown, and its display exceeds selectivity 5 orders of magnitude to RI isotype to the selectivity of the RII isotype of PKA.Underlined residue represents the position of the amino acid replacement relative to AKAP-IS sequence, the combination of the DDD part of described alternative increase and RII α.In the sequence, N-terminal Q residue is numbered as residue numbers 4 and C-terminal A residue is residue numbers 20.Wherein can to carry out substituting with impact the residue of the avidity of RII α as residue 8,11,15,16,18,19 and 20 people such as (, 2006) Gold.Expection is in some selectable embodiment, and available SuperAKAP-IS sequence replacing AKAP-ISAD partial sequence is to prepare dual specific immune cell factor DNL construct.Other selectable sequence that can be used to alternative AKAP-ISAD sequence is shown in hereinafter.Underline substituting relative to AKAP-IS sequence.Expection, about AKAP-IS sequence, AD part also can comprise extra N-terminal residue halfcystine and glycine and C-terminal residue glycine and halfcystine.

SuperAKAP-IS

QIEY VAKQIVD YAI HQA(SEQIDNO:24)

Selectable AKAP sequence

QIEY KAKQIVD HAI HQA(SEQIDNO:25)

QIEY HAKQIVD HAI HQA(SEQIDNO:26)

QIEY VAKQIVD HAI HQA(SEQIDNO:27)

Fig. 2 of the people such as Gold discloses the extra DDD-binding sequence from multiple AKAP albumen hereinafter shown.

rII-specificity AKAP

AKAP-KL

PLEYQAGLLVQNAIQQAI(SEQIDNO:28)

AKAP79

LLIETASSLVKNAIQLSI(SEQIDNO:29)

AKAP-Lbc

LIEEAASRIVDAVIEQVK(SEQIDNO:30)

rI-specificity AKAP

AKAPce

ALYQFADRFSELVISEAL(SEQIDNO:31)

RIAD

LEQVANQLADQIIKEAT(SEQIDNO:32)

PV38

FEELAWKIAKMIWSDVF(SEQIDNO:33)

dual-specificity AKAP

AKAP7

ELVRLSKRLVENAVLKAV(SEQIDNO:34)

MAP2D

TAEEVSARIVQVVTAEAV(SEQIDNO:35)

DAKAP1

QIKQAAFQLISQVILEAT(SEQIDNO:36)

DAKAP2

LAWKIAKMIVSDVMQQ(SEQIDNO:37)

The people such as Stokka (2006) also developed the peptide competitor of the AKAP in conjunction with PKA hereinafter shown.Described peptide antagonists is named as Ht31, RIAD and PV-38.The avidity of the RII isotype to PKA that the display of Ht-31 peptide is larger, and the avidity to RI that RIAD and V-38 display is larger.

Ht31

DLIEEAASRIVDAVIEQVKAAGAY(SEQIDNO:38)

RIAD

LEQYANQLADQIIKEATE(SEQIDNO:39)

PV-38

FEELAWKIAKMIWSDVFQQC(SEQIDNO:40)

The people such as Hundsrucker (2006) also developed other peptide competitor of the AKAP in conjunction with PKA, have the binding constant of the DDD of the RII form to PKA being low to moderate 0.4nM.The sequence of multiple AKAP antagonistic peptides is provided in the table 1 of the people such as the Hundsrucker hereinafter copied.

Table 1AKAP peptide sequence

AKAPIS represents synthesis RII subunit binding peptide.Other peptides all are derived from the RII binding domains of shown AKAP.

Peptide sequence

AKAPISQIEYLAKQIVDNAIQQA(SEQIDNO：I5)

AKAPIS-PQIEYLAKQIPDNAIQQA(SEQIDNO：41)

Ht31KGADLIEEAASRIVDAVIEQVKAAG(SEQIDNO：42)

Ht31-PKGADLIEEAASRIPDAPIEQVKAAG(SEQIDNO：43)

AKAP7δ-wt-pepPEDAELVRLSKRLVENAVLKAVQQY(SEQIDNO：44)

AKAP7δ-L304T-pepPEDAELVRTSKRLVENAVLKAVQQY(SEQIDNO：45)

AKAP7δ-L308D-pepPEDAELVRLSKRDVENAVLKAVQQY(SEQIDNO：46)

AKAP7δ-P-pepPEDAELVRLSKRLPENAVLKAVQQY(SEQIDNO：47)

AKAP7δ-PP-pepPEDAELVRLSKRLPENAPLKAVQQY(SEQIDNO：48)

AKAP7δ-L314E-pepPEDAELVRLSKRLVENAVEKAVQQY(SEQIDNO：49)

AKAP1-pepEEGLDRNEEIKRAAFQIISQVISEA(SEQIDNO：50)

AKAP2-pepLVDDPLEYQAGLLVQNAIQQAIAEQ(SEQIDNO：51)

AKAP5-pepQYETLLIETASSLVKNAIQLSIEQL(SEQIDNO：52)

AKAP9-pepLEKQYQEQLEEEVAKVIVSMSIAFA(SEQIDNO：53)

AKAP10-pepNTDEAQEELAWKIAKMIVSDIMQQA(SEQIDNO：54)

AKAP11-pepVNLDKKAVLAEKIVAEAIEKAEREL(SEQIDNO：55)

AKAP12-pepNGILELETKSSKLVQNIIQTAVDQF(SEQIDNO：56)

AKAP14-pepTQDKNYEDELTQVALALVEDVINYA(SEQIDNO：57)

Rab32-pepETSAKDNINIEEAARFLVEKILVNH(SEQIDNO：58)

At the conservative residue of the AD structural domain camber of different AKAP albumen by reference to following AKAPIS sequence hereinafter by underlining sign.Except the interpolation of C-terminal alanine residue, described residue with observed by the people such as Alto (2003) identical.(see Fig. 4 of the people such as Hundsrucker (2006), described document being incorporated to by reference herein).The sequence with the peptide antagonists of the extra high avidity to RIIDDD sequence is the sequence of AKAP-IS, AKAP7 δ-wt-pep, AKAP7 δ-L304T-pep and AKAP7 δ-L308D-pep.

AKAP-IS

QIEYL AKQ IVDN AIQQ A(SEQIDNO:15)

The people such as Carr (2001) examine different AKAP from people and non-human protein's matter in conjunction with the sequence homology degree between DDD sequence and identify in DDD sequence and seem the residue that topnotch is conservative in different DDD part.These by reference men and women PKARII α DDD sequence hereinafter by underlining sign.Residue conservative is especially indicated by italics further.Described residue with proposed by the people such as Kinderman (2006) overlapping for those residues important in conjunction with AKAP albumen, but not identical with it.Therefore, hereinafter indicate potential DDD sequence, wherein " X " represents that conserved amino acid substitutes.

S HIQ IPP GLT ELLQGYTV EVLRQ QPP DLVEFAVE YFTR LR EA RA(SEQIDNO:13)

X HIX IPX GLX ELLQGYTX EVLRX QPX DLVEFAXX YFXX LX EX RX(SEQIDNO:59)

Those skilled in the art will understand that, generally speaking, these are preferably can keep constant residue in generation amino acid replacement at the amino-acid residue that DDD and the AD sequence camber from different proteins is guarded, but not too the residue of high conservative can more easily be changed to produce the sequence variants of AD and/or DDD sequence described herein.

Except DDD and/or AD part sequence variants except, in certain embodiments, can preferably antibody moiety or connection antibody and AD sequence linker peptide sequence in calling sequence make a variation.In an illustrative example, 3 of fusion rotein sequence may below variants be shown in.

(L)

QKSLSLSPGLGSGGGGSGGCG(SEQIDNO:60)

(A)

QKSLSLSPGAGSGGGGSGGCG(SEQIDNO:61)

(-)

QKSLSLSPGGSGGGGSGGCG(SEQIDNO:62)

Amino acid replacement

In certain embodiments, disclosed method and composition can comprise and has one or more protein of alternative amino-acid residue or the generation of peptide and use.Optimization natural antibody, chimeric antibody, the structure of humanized antibody or people's antibody or AD or DDD sequence, physics and/or treatment feature is come by replacing one or more amino-acid residue.Such as, as everyone knows by improving the functional character of humanized antibody with corresponding FR amino acid replacement a limited number of people framework region (FR) amino acid of parental generation murine antibody.When framework region amino acid residue and CDR residue very near time, this situation is especially correct.

In other cases, by the treatment characteristic of a limited number of amino acid replacement optimization antibody, such as to the dissociation of the binding affinity of target antigen, antibody and its target antigen or dissociation rate or even antibody to the efficiency of the induction of CDC (complement dependent cytotoxicity) or ADCC (antibody dependent cellular cytotoxicity).

In selectable embodiment, can further optimization for generation of DDD and/or the AD sequence of DNL construct of the present invention, such as, to increase DDD-AD binding affinity.Above discuss the potential sequence variations in DDD or AD sequence.

Those skilled in the art will appreciate that generally speaking, amino acid replacement generally includes with the another kind of amino acid replacement amino acid with relatively similar character (that is, conserved amino acid substitutes).The impact on protein structure and function of the character of different aminoacids and amino acid replacement is the theme of extensively research and knowledge in this area all the time.

Such as, amino acid whose hydrophilic index (Kyte & Doolittle, 1982, J.Mol.Biol., 157:105-132) can be considered.Amino acid whose relative hydropathic character facilitates gained Secondary structure, and this determines the interaction of protein and other molecule conversely.Each is amino acid based is endowed hydrophilic index (Kyte & Doolittle, 1982) in its hydrophobicity and charge characteristic, and these amino acid are: Isoleucine (+4.5); α-amino-isovaleric acid (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Halfcystine/Gelucystine (+2.5); Methionine(Met) (+1.9); L-Ala (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophane (-0.9); Tyrosine (-1.3); Proline(Pro) (-1.6); Histidine (-3.2); L-glutamic acid (-3.5); Glutamine (-3.5); Aspartic acid (-3.5); L-asparagine (-3.5); Methionin (-3.9); With arginine (-4.5).Substitute generation is conservative, the amino acid whose use of its hydrophilic index in ± 2 is preferred, and its hydrophilic index is preferred in ± 1, and its hydrophilic index is preferred further in ± 0.5.

Amino acid replacement also can consider the wetting ability (such as, the 4th, 554, No. 101 United States Patent (USP)s) of amino-acid residue.Hydrophilicity value is given to amino-acid residue: arginine (+3.0); Methionin (+3.0); Aspartic acid (+3.0); L-glutamic acid (+3.0); Serine (+0.3); L-asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline(Pro) (-0.5.+-.1); L-Ala (-0.5); Histidine (-0.5); Halfcystine (-1.0); Methionine(Met) (-1.3); α-amino-isovaleric acid (-1.5); Leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophane (-3.4).It is preferred for carrying out alternative amino acid with other amino acid with similar hydropathic.

Other considers the size comprising amino acid side chain.Such as, substituting the amino acid such as glycine or Serine with compact side chain with the amino acid such as tryptophane or tyrosine with bulky side chain is not preferred usually.Also the impact of different aminoacids residue on secondary protein structure will be considered.Pass through empirical studies, different aminoacids residue on protein domain take the impact of tendency of alpha-helix, beta-pleated sheet or reversion secondary structure to be determined and to be in this area to be known (see, such as, Chou & Fasman, 1974, Biochemistry, 13:222-245; 1978, Ann.Rev.Biochem., 47:251-276; 1979, Biophys.J., 26:367-384).

Based on such consideration and empirical studies widely, the expression that conserved amino acid substitutes has been fabricated and has been known in this area.Such as, arginine and Methionin; L-glutamic acid and aspartic acid; Serine and Threonine; Glutamine and l-asparagine; And α-amino-isovaleric acid, leucine and Isoleucine.Selectively: Ala (A) leu, ile, val; Arg (R) gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys (C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H) asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met, ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F) leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W) phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other consideration for amino acid replacement comprises residue and whether is positioned at the inside of protein or whether is exposed to solvent.For internal residues, conservative substituting can comprise: 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, such as, the PROWL website on rockefeller.edu).For the residue being exposed to solvent, conservative substituting can comprise: 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. (Id.).Build multiple matrix to help select amino acid replacement, such as PAM250 rating matrix, Dayhoff matrix, Grantham matrix, McLachlan matrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix, Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.).

In mensuration amino acid replacement, also existence that is intermolecular or intramolecular bond can be considered, such as positively charged residue (such as, His, Arg, Lys) and electronegative residue (such as, Asp, Glu) between the formation of disulfide linkage between the formation of ionic linkage or neighbouring cysteine residues.

Other amino acid whose method any in the protein sequence of encoding with any amino acid replacement is known and only normal experiment to those skilled in the art, such as by the technology of site-directed mutagenesis or by synthesizing and assemble the oligonucleotide that coded amino acid substitutes, then spliced and carried out into expression vector constructs.

Cytokine and other immunomodulator

In certain preferred aspects, effector part can be immunomodulator.Immunomodulator is the immune reagent changing, suppress or stimulate health when it is present.The immunomodulator used can comprise cytokine, stem cell factor, lymphotoxin, Hemopoietic factor, G CFS (CSF), Interferon, rabbit (IFN), erythropoietin, thrombopoietin and combination thereof.Useful especially is the stem cell factor of lymphotoxin such as tumour necrosis factor (TNF), Hemopoietic factor such as interleukin-(IL), G CFS such as granulocyte colony-stimulating factor (G-CSF) or rHuGM-CSF (GM-CSF), Interferon, rabbit such as interferon-' alpha ' ,-β or-γ and stem cell factor such as called after " the S1 factor ".

In certain preferred aspects, effector part is cytokine such as lymphokine, Monokines, somatomedin and conventional polypeptide hormone.Cytokine comprises: tethelin, as human growth hormone, N-methionyl human growth hormone and Trobest; Rat parathyroid hormone 1-34; Thyroxine; Regular Insulin; Proinsulin; Relaxin; Relaxation precipitinogen; Glycoprotein hormones, such as follicular stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and lutropin (LH); Placenta growth factor (PIGF), pHGF; Prostaglandin(PG), fibroblast growth factor; Prolactin; Galactagogin, OB albumen; Tumor necrosis factor-alpha and-β; Mullerian inhibiting substance (mullerianinhibitingsubstance); Mouse gonad-stimulating hormone related peptides; Statin; Activator; Vascular endothelial growth factor; Integrin; Thrombopoietin (TPO); Nerve growth factor is NGF-β such as; PDGF; Transforming growth factor (TGF), as TGF-α and TGF-β; Insulin like growth factor-1 and-II; Erythropoietin (EPO); Bone-inducing factor; Interferon, rabbit is interferon-' alpha ' ,-β and-γ such as; G CFS (CSF) is scavenger cell-CSF (M-CSF) such as; Interleukin (II) 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, tumour necrosis factor (TNF, such as TNF-α) and LT.

The aminoacid sequence of protein or peptide immunomodulator such as cytokine is well known in the art and this type of known sequence any can be used for implementing the present invention.Those skilled in the art will know that numerous sources of the Public information about cytokine sequence.Such as, ncbi database comprises protein and the nucleic acid sequence encoding of a large amount of cytokine and immunomodulator, and described cytokine and immunomodulator are such as erythropoietin (GenBankNM000799), IL-1 β (GenPeptAAH08678), GM-CSF (GenPeptAAA52578), TNF-α (GenPeptCAA26669), interferon-' alpha ' (GenPeptAAA52716.1), interferon-' alpha ' 2b (GenPeptAAP20099.1) and above listed in fact any peptide or Western Immuno conditioning agent.The qualification substantially suitable amino acid of any target protein matter or peptide effector part and/or nucleotide sequence is conventional thing to those skilled in the art.

Antibody and antibody fragment

Technology for the preparation of the monoclonal antibody of in fact anti-any target antigen is well known in the art.See, such as, Kohler and Milstein, Nature256:495 (1975), and people's (volume) such as Coligan, CURRENTPROTOCOLSINIMMUNOLOGY, 1st volume, 2.5.1-2.6.7 page (JohnWiley & Sons1991).In brief, by obtaining monoclonal antibody as follows: with the composition injection mouse comprising antigen, take out spleen to obtain bone-marrow-derived lymphocyte, by described bone-marrow-derived lymphocyte and myeloma cell fusion to produce hybridoma, clone hybridization knurl, select to produce the clone for the antibody of described antigen, cultivate and produce for the clone of the antibody of described antigen and from Hybridoma culture separation antibody.

Multiple perfect technology can be utilized from Hybridoma culture abstraction and purification MAb.This type of isolation technique comprises the affinity chromatography, size exclusion chromatography and the ion exchange chromatography that utilize Protein A sepharose.See, such as, the Coligan of 2.7.1-2.7.12 page and 2.9.1-2.9.3 page.In addition, see people such as Baines, " PurificationofImmunoglobulinG (IgG), " inMETHODSINMOLECULARBIOLOGY, the 10th volume, 79-104 page (TheHumanaPress, Inc.1992).

Initially producing after for immunogenic antibody, the sequence of antibody can be measured, be prepared by recombinant technology subsequently.Humanization and the sign of murine antibody and antibody fragment are known to those skilled in the art.Use derived from the antibody component of humanized antibody, chimeric antibody or people's antibody eliminates the potential problems relevant to the immunogenicity of mouse constant region.

Chimeric antibody

Chimeric antibody is recombinant protein, and wherein the variable region of people's antibody is replaced by the variable region such as comprising the mouse antibodies of the complementarity-determining region (CDR) of mouse antibodies.When using to experimenter, chimeric antibody demonstrates immunogenicity to be reduced and stability increase.General technology for cloning murine immunoglobulin variable domains is disclosed in the people such as such as Orlandi, in Proc.Nat'lAcad.Sci.USA86:3833 (1989).Known to those skilled in the art for building the technology of embedding antibody.As an example, the people such as Leung, Hybridoma13:469 (1994) is by the V of mouse LL2 (anti-CD22 monoclonal antibody) of encoding _κand V _hthe DNA sequence dna of structural domain and point others κ and IgG ₁constant region domain combination produces LL2 chimeric antibody.

Humanized antibody

Technology for generation of humanization MAb be known in the art (see, such as, the people such as Jones, Nature321:522 (1986), the people such as Riechmann, Nature332:323 (1988), the people such as Verhoeyen, Science239:1534 (1988), the people such as Carter, Proc.Nat'lAcad.Sci.USA89:4285 (1992), Sandhu, the people such as Crit.Rev.Biotech.12:437 (1992) and Singer, J.Immun.150:2844 (1993)).By mouse CDR is carried out the chimeric or mouse monoclonal antibody of humanization from the corresponding variable domains that the variable heavy chain of mouse immuning ball protein and variable light are transferred to people's antibody.Mouse framework district (FR) in chimeric mAb also employment FR sequence replaces.Usually cause the minimizing of affinity of antibody owing to being transferred in people FR by mouse CDR simply or even losing, the original avidity therefore in order to recover murine antibody may need extra modification.This is by realizing the antibody that their mouse counterpart substitutes to obtain to its epi-position has good combination avidity of the one or more people's residues in FR district.See, the people such as such as Tempest, the people such as Biotechnology9:266 (1991) and Verhoeyen, Science239:1534 (1988).Usually, different with their mouse counterpart and to be tightly positioned at or those people FR amino-acid residues of contacting one or more cdr amino acid residue can be alternative candidates.

People's antibody

(such as, the people such as Mancini, 2004, NewMicrobiol.27:315-28 known in the art by combined method or through the method that the transgenic animal that human immunoglobulin gene's seat transforms produce complete human antibody; Conrad and Scheller, 2005, Comb.Chem.HighThroughputScreen.8:117-26; Brekke and Loset, 2003, Curr.Opin.Phamacol.3:544-50).Also can utilize gene or chromosomal transfection method and phage display technology (all described technology are all known in this area) structure complete human antibody.See people such as such as McCafferty, Nature348:552-553 (1990).Such complete human antibody's expection shows than chimeric antibody or the less side effect of humanized antibody and works in vivo as substantially endogenous people's antibody.In certain embodiments, claimed Method and Process can use the people's antibody produced by this type of technology.

In an alternative embodiment, phage display technology can be used for producing people's antibody (such as, Dantas-Barbosa etc., 2005, Genet.Mol.Res.4:126-40).People's antibody from normal people or can show particular disease states as prepared (Dantas-Barbosa etc., 2005) the people of cancer.The favourable aspect building people's antibody from diseased individuals is that the full storehouse of circulating antibody may have Preference to the antibody for Disease associated antigens.

In a limiting examples of this method, Dantas-Barbosa etc. (2005) construct the Phage displaying library of the human Fab's antibody fragment from Patients with Osteosarcoma.Generally speaking, total serum IgE is available from CBL (Id.).From the full storehouse of μ, γ and κ chain antibody, clone recombinant Fab, and be inserted into Phage displaying library (Id.).RNA is converted into cDNA by using to heavy chain and the sequence-specific primer of light chain immunoglobulins, and for the preparation of FabcDNA library people such as (, 1991, J.Mol.Biol.222:581-97) Marks.Library construction press Andris-Widhopf etc. (2000, at PhageDisplayLaboratoryManual, Barbas etc. (writing), 1st edition, in ColdSpringHarborLaboratoryPress, ColdSpringHarbor, NYpp.9.1 to 9.22) carry out.Final Fab fragment restriction endonuclease digests, and then inserts phage genome to prepare described Phage displaying library.This type of library can use standard bacteriophage display packing known in the art carry out screening (see, such as, PasqualiniandRuoslahti, 1996, Nature380:364-366; Pasqualini, 1999, TheQuart.J.Nucl.Med.43:159-162).

Phage display can be implemented in a variety of forms, about their summary, see such as Johnson and Chiswell, CurrentOpinioninStructuralBiology3:5564-571 (1993).The B cell of Activated in Vitro also can be utilized to produce people's antibody.See, the 5th, 567, No. 610 and the 5th, 229, No. 275 United States Patent (USP)s, be incorporated herein by reference in their entirety described patent.It will be understood by those skilled in the art that these technology be exemplary and can utilize for the preparation of with sieve human antibodies or any currently known methods of antibody fragment.

In another alternate embodiment, use the immunization experiment scheme of standard, can be used for the antibody of generation for any immune target substantially through genetic engineering modified with the transgenic animal producing people's antibody.For obtaining the method for people's antibody from transgenic animal by people such as Green, NatureGenet.7:13 (1994), the people such as Lonberg, the people such as Nature368:856 (1994) and Taylor, Int.Immun.6:579 (1994) is open.The non-limiting example of this system is Abgenix's (Fremont, CA) (such as, the people such as Green, 1999, J.Immunol.Methods231:11-23).? with in similar animal, mouse antibody gene by inactivation, and replaces with functional human antibody gene, but the immune remainder of mouse still keeps complete.

transformed the YAC (yeast artificial chromosome) of germline configuration, this YAC comprises groups of people IgH and Ig kappa gene seat, comprises most of region of variable region sequences, and auxiliary gene and regulating and controlling sequence.People variable region Quan Ku can be used for preparing the B cell producing antibody, and B cell merges to hybridoma by known technology.With target antigen immunity to produce people's antibody by normal immune response, described people's antibody is undertaken gathering in the crops and/or producing by above-mentioned standard technique.There is multiple germline available, each germline all can produce different classes of antibody.People's antibody that transgenosis produces has demonstrated treatment potentiality, and remains the pharmacokinetic properties (Green etc., 1999) of normal people's antibody.It will be understood by those skilled in the art that claimed composition and method are not limited to use system, but can use through the genetic engineering modified any transgenic animal producing people's antibody.

Antibody fragment

Known technology can be utilized to produce the antibody fragment identifying defined epitope.Antibody fragment is the antigen-binding portion thereof such as F (ab') of antibody ₂, Fab', F (ab) ₂, Fab, Fv, sFv etc.F (ab ') is produced by Pepsin degradation antibody molecule ₂fragment, and by reduction F (ab ') ₂the disulphide bridges of fragment produces Fab ' fragment.Selectively, Fab ' expression library (people such as Huse, 1989, Science, 246:1274-1281) can be built to allow fast and easily qualification has the specific mono-clonal Fab ' fragment of expectation.F (ab) is produced by Papain digestion of antibodies ₂fragment, and obtain Fab fragment by disulfide reduction.

Single Chain Fv Molecule A (scFv) comprises VL structural domain and VH structural domain.VL and VH structural domain associates to form target combining site.These two structural domains are covalently bound further by peptide linker (L).The 4th is disclosed in for the preparation of scFv molecule and the method designing suitable peptide linker, 704, No. 692 United States Patent (USP)s, the 4th, 946, No. 778 United States Patent (USP)s, R.Raag and M.Whitlow, " SingleChainFvs. " FASEB the 9th volume: 73-80 (1995) and R.E.Bird and B.W.Walker, " SingleChainAntibodyVariableRegions; " TIBTECH, in the 9th volume: 132-137 (1991).

Technology for generation of single domain antibody (DAB) is also known in this area, disclosed in the people (2006, ProtExpressPurif51:253-259) such as such as Cossins (being incorporated to by reference herein).

Dispersal risk fragment is carried out by proteolysis full length antibody or by the DNA expressing the described fragment of coding intestinal bacteria (E.coli) or another kind of Su Zuzhong.Ordinary method can be utilized to obtain antibody fragment by stomach en-or Papain enzyme liberating full length antibody.These class methods are by such as Goldenberg, and the 4th, 036, No. 945 and the 4th, 331, No. 647 United States Patent (USP)s and the bibliography comprised herein describe.In addition, see people such as Nisonoff, ArchBiochem.Biophys.89:230 (1960); Porter, Biochem.J.73:119 (1959), the people such as Edelman, inMETHODSINENZYMOLOGY the 1st volume, the Coligan of the 422nd page (AcademicPress1967) and 2.8.1-2.8.10 and 2.10.-2.10.4 page.

Known antibodies

The antibody used can be commercially available from many known sources.Such as, multiple antibody-secreting type hybridoma strain can available from American type culture collection (ATCC, Manassas, VA).The antibody of a large amount of anti-various disease target (including but not limited to tumor associated antigen) has been deposited in ATCC and/or has had the variable region sequences of announcement, and can obtain for claimed method and composition.See, such as, the 7th, 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; 6,720,15; 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, No. 338 United States Patent (USP)s.These are only that exemplary and many other antibody of kind and their hybridoma are known in the art.It will be understood by those skilled in the art that antibody sequence or the antibody-secreting type hybridoma of anti-almost any Disease associated antigens are searched for ATCC, NCBI and/or USPTO database to obtain by just resisting the antibody of selected disease-related target simply.The antigen-binding domains of the antibody that standard art amplification well known in the art can be used to clone, is cut, is then connected into expression vector, is transferred to suitable host cell, and for protein production.

Immunoconjugates

In certain embodiments, antibody or its fragment can be conjugated to one or more therapeutical agents or diagnostic reagent.Therapeutical agent need not be identical but can be different, such as medicine and radio isotope.Such as, can be by ¹³¹i mixes the tyrosine of antibody or fusion rotein and is connected to the medicine of ε amino of lysine residue.Also therapeutical agent and diagnostic reagent can be connected to such as reduced form SH group and/or carbohydrate side chains.Many methods for the preparation of the covalently or non-covalently conjugate of therapeutical agent or diagnostic reagent and antibody or fusion rotein are known in the art and can utilize any method known like this.

By the hinge area being formed in reduced form antibody component of disulfide linkage connects therapeutical agent or diagnostic reagent.Selectively, heterobifunctional crosslinker such as N-succinyl 3-(2-pyridine dithio) propionic ester (SPDP) can be used to connect this type of reagent.The people such as Yu, Int.J.Cancer56:244 (1994).Be well known in the art for such general technology puted together.See, such as, Wong, CHEMISTRYOFPROTEINCONJUGATIONANDCROSS-LINKING (CRCPress1991); The people such as Upeslacis, people's (volume) such as " ModificationofAntibodiesbyChemicalMethods, " inMONOCLONALANTIBODIES:PRINCIPLESANDAPPLICATIONS, Birch, 187-230 page (Wiley-Liss, Inc.1995); Price, " ProductionandCharacterizationofSyntheticPeptide-DerivedA ntibodies; " inMONOCLONALANTIBODIES:PRODUCTION, ENGINEERINGANDCLINICALAPPLICATION, the people such as Ritter (volume), 60-84 page (CambridgeUniversityPress1995).Selectively, therapeutical agent or diagnostic reagent is puted together by the carbohydrate fraction in the Fc district of antibody.Glycosyl can be used for the loading increasing the identical reagent combined with sulfydryl, or carbohydrate fraction can be used in conjunction with different therapeutical agents or diagnostic reagent.

Method for peptide is conjugated to antibody component via the carbohydrate fraction of antibody is known to those skilled in the art.See, such as, the people such as Shih, Int.J.Cancer41:832 (1988); The people such as Shih, the people such as Int.J.Cancer46:1101 (1990) and Shih, U.S.PatentNo.5,057,313, described document is incorporated herein by reference in their entirety.General method comprises the antibody component making to have oxidized form carbohydrate fraction and reacts with the carrier polymer with at least one unhindered amina function.This reaction causes producing initial schiff bases (imines) bonding, and it is stable to obtain to form final conjugate by being reduced into secondary amine.

If the antibody being used as the antibody component of immunoconjugates is antibody fragment, so Fc district can not exist.But, carbohydrate fraction may be introduced the variable region of light chain of full length antibody or antibody fragment.See, such as, the people such as Leung, J.Immunol.154:5919 (1995); The people such as Hansen, U.S.PatentNo.5, the people such as 443,953 (1995), Leung, the 6th, 254, No. 868 United States Patent (USP)s, are incorporated herein by reference in their entirety described document and patent.Genetic engineering modified carbohydrate fraction is used for connecting therapeutical agent or diagnostic reagent.

In certain embodiments, sequestrant can be connected to antibody, antibody fragment or fusion rotein, then for chelating therapy agent or diagnostic reagent such as radionuclide.Exemplary chelators includes but not limited to DTPA (such as Mx-DTPA), DOTA, TETA, NETA or NOTA.For sequestrant put together and use its metal or other part be connected to method of protein be well known in the art (see, such as, the 12/112nd, No. 289 U.S. Patent applications, are incorporated herein by reference in their entirety).

In certain embodiments, by radioactive metal or paramagnetic ion being connected to protein or peptide with the reagent react with long-tail, multiple chelation group for coupled ion can be connected to described long-tail.Such tail can be polymkeric substance such as poly-lysine, polysaccharide or other have side base derivative polymer or can derivative chain, described side base can in conjunction with the known chelation group that can be used for this purposes, such as ethylenediamine tetraacetic acid (EDTA) (EDTA), diethylene triamine pentacetic acid (DTPA) (DTPA), porphyrin, polyamines, crown ether, two-thiosemicarbazone, poly-oxime (polyoxime) etc.

Sequestrant can be connected directly to antibody or peptide, such as, disclosed in the 4th, 824, No. 659 United States Patent (USP)s (it is incorporated to herein by reference).The combination of useful especially metal-chelant comprise with the diagnosis isotropic substance for radiological imaging (radioimaging) in the general energy region of 60 to 4,000keV (such as ¹²⁵i, ¹³¹i, ¹²³i, ¹²⁴i, ⁶²cu, ⁶⁴cu, ¹⁸f, ¹¹¹in, ⁶⁷ga, ⁶⁸ga, ^99mtc, ^94mtc, ¹¹c, ¹³n, ¹⁵o, ⁷⁶br) the 2-benzyl-DTPA used together and monomethyl thereof and cyclohexyl analogs.When with non-radioactive metal such as manganese, iron and gadolinium complexing time, identical sequestrant can be used for MRI.Macrocyclic chelants, as NOTA, DOTA and TETA, can be used for various metals and radiometal, is particularly respectively used to gallium, yttrium and copper radionuclide.By the size according to metal target regulating ring, this type of metal-chelant complex can be made highly stable.Can comprise the sequestrant of other lopps type, such as Macrocyclic polyester, it is concerned for stably in conjunction with nucleic such as ²²³ra (for RAIT).

Recently, disclosed for PET scanning technique ¹⁸the method of F-mark, such as, undertaken by the reaction of F-18 and metal or other atom such as aluminium.Can be by ¹⁸such as DOTA, NOTA or NETA complexing of F-Al conjugate and chelation group, described chelate group is connected directly to antibody or is used to mark in pre-targeting method can the construct of target.Such F-18 labeling technique is disclosed in the 7th, 563, in 433 United States Patent (USP)s, embodiment part is incorporated to by reference herein.

Therapeutical agent

In selectable embodiment, therapeutical agent can be used, be conjugated to DNL mixture of the present invention, or before antibody is used, simultaneously or use it dividually afterwards, described therapeutical agent is such as cytotoxic agent, anti-angiogenic agent, short apoptosis agent, microbiotic, hormone, hormone antagonist, chemokine, medicine, prodrug, toxin, enzyme or other reagent.The medicine used can have the medicinal property being selected from antimitotic agent, antikinase, alkylating agent, metabolic antagonist, microbiotic, alkaloid, anti-angiogenic agent, short apoptosis agent and its combination.

The illustrative drug used can comprise 5 FU 5 fluorouracil, aplidin, azaribine, Anastrozole, anthracycline, bendamustine, bleomycin, Velcade, bryostatin 1, busulfan, calicheamicin, camptothecine, carboplatin, 10-hydroxycamptothecine, carmustine, celecoxib, Chlorambucil, cis-platinum (CDDP), cox 2 inhibitor, irinotecan (CPT-11), SN-38, carboplatin, CldAdo, camptothecine, endoxan, cytosine arabinoside, Dacarbazine, docetaxel, dactinomycin, daunorubicin, Dx, 2-pyrrolin Dx (2P-DOX), cyano-morpholine is for Dx, glucuronide Dx, glucuronide epirubicin, estramustine, epipodophyllotoxin, estrogen receptor binding agents, Etoposide (VP16), glucuronide Etoposide, etoposide phosphate, floxuridine (FUdR), 3', 5'-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, farnesyl protein converting enzyme inhibitor, gemcitabine, hydroxyurea, idarubicin, ifosfamide, L-Asnase, Revlimid, Calciumlevofolinate, lomustine, mustargen, melphalan, mercaptopurine, Ismipur, methotrexate, mitoxantrone, Plicamycin, mitomycin, mitotane, nvelbine, nitrourea, Plicamycin (plicomycin), Procarbazine, taxol, pentostatin, PSI-341, raloxifene, semustine, streptozocin (streptozocin), tamoxifen, PTX, Temozolomide (the moisture form of DTIC), anti-platinum (transplatinum), Thalidomide, Tioguanine, phosphinothioylidynetrisaziridine, teniposide, Hycamtin, Uramustine, vinorelbine, vinealeucoblastine(VLB), vincristine(VCR) and vinca alkaloids.

The toxin used can comprise Ricin, abrin, alpha toxin, saporin, rnase (RNA enzyme), such as ranpirnase, DNA enzymatic I, staphylococcal enterotoxin A, Pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin and pseudomonas intracellular toxin.

The chemokine used can comprise RANTES, MCAF, MIP1-α, MIP1-β and IP-10.

In certain embodiments, anti-angiogenic agent such as angiostatin (angiostatin) can be used, baculostatin, canstatin (canstatin), mammary gland silk presses down albumen (maspin), VEGF antibody, anti-PlGF peptide and antibody, anti-angiogenesis factor antibody, anti-Flk-1 antibody, anti-Flt-1 antibody and peptide, anti-Kras antibody, anti-cMET antibody, anti-MIF (macrophage migration-supressor) antibody, ln peptide, fibronectin peptide, Type 1 plasminogen activator inhibitor, tissue inhibitor of metalloproteinase, Interferon, rabbit, interleukin 12, IP-10, Gro-β, thrombospondin, 2ME2, proliferin associated protein, carboxylic acid amides triazole (carboxiamidotriazole), CM101, Marimastat (Marimastat), the many vitriol of piperylene, angiopoietin-2, interferon alpha, Antibiotic TAN 420F, PNU145156E, 16K prolactin fragments, Linomide (linomide), Thalidomide, pentoxifylline, genistein, TNP-470, endostatin, taxol, accutin, angiostatin, cidofovir, vincristine(VCR), bleomycin, AGM-1470, platelet factor 4 or Minocycline HCl.

The immunomodulator used can be selected from cytokine, stem cell factor, lymphotoxin, Hemopoietic factor, G CFS (CSF), Interferon, rabbit (IFN), erythropoietin, thrombopoietin and its combination.Useful especially is the stem cell factor of lymphotoxin such as tumour necrosis factor (TNF), Hemopoietic factor such as interleukin-(IL), G CFS such as G-CSF (G-CSF) or CM-CSF (GM-CSF), Interferon, rabbit such as interferon-' alpha ' ,-β or-γ and stem cell factor such as called after " the S1 factor ".Cytokine comprises tethelin such as human growth hormone, N-methionyl human growth hormone and Trobest; Rat parathyroid hormone 1-34; Thyroxine; Regular Insulin; Proinsulin; Relaxin; Relaxation precipitinogen; Glycoprotein hormones is follicular stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and lutropin (LH) such as; PHGF; Prostaglandin(PG), fibroblast growth factor; Prolactin; Galactagogin, OB albumen; Tumor necrosis factor-alpha and-β; Mullerian inhibiting substance (mullerianinhibitingsubstance); Mouse gonad-stimulating hormone related peptides; Statin; Activator; Vascular endothelial growth factor; Integrin; Thrombopoietin (TPO); Nerve growth factor is NGF-β such as; PDGF; Transforming growth factor (TGF) such as TGF-α and TGF-β; Insulin like growth factor-1 and-II; Erythropoietin (EP0); Bone-inducing factor; Interferon, rabbit is interferon-' alpha ' ,-β and-γ such as; G CFS (CSF) is scavenger cell-CSF (M-CSF) such as; Interleukin-(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, tumour necrosis factor and LT.

The radionuclide used includes but not limited to ¹¹¹in, ¹⁷⁷lu, ²¹²bi, ²¹³bi, ²¹¹at, ⁶²cu, ⁶⁷cu, ⁹⁰y, ¹²⁵i, ¹³¹i, ³²p, ³³p, ⁴⁷sc, ¹¹¹ag, ⁶⁷ga, ¹⁴²pr, ¹⁵³sm, ¹⁶¹tb, ¹⁶⁶dy, ¹⁶⁶ho, ¹⁸⁶re, ¹⁸⁸re, ¹⁸⁹re, ²¹²pb, ²²³ra, ²²⁵ac, ⁵⁹fe, ⁷⁵se, ⁷⁷as, ⁸⁹sr, ⁹⁹mo, ¹⁰⁵rh, ¹⁰⁹pd, ¹⁴³pr, ¹⁴⁹pm, ¹⁶⁹er, ¹⁹⁴ir, ¹⁹⁸au, ¹⁹⁹au and ²¹¹pb.Therapeutic radionuclides preferably has 60 to 6, the decay energy within the scope of 000keV, and the decay of Auger radiator can be preferably in 60 to 200keV scope, the decay of beta emitter can be preferably 100-2,500keV, and the decay of alpha emitter can be preferably 4,000-6,000keV.The maximum decay of useful beta-radiation nucleic can be preferably 20-5,000keV, is more preferably 100-4,000keV, is most preferably 500-2,500keV.The radionuclide carrying out decaying basically by radiation Auger particle is also preferred.Such as, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119,1-125, Ho-161, Os-189m and Ir-192.The decay of useful beta-radiation nucleic can be preferably <1,000keV, is more preferably <100keV, is most preferably <70keV.The radionuclide carrying out decaying basically by generation alpha-particle is also preferred.This type of radionuclide includes but not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213 and Fm-255.The decay of the radionuclide of useful alpha-radiation can be preferably 2,000-10,000keV, is more preferably 3,000-8,000keV, is most preferably 4,000-7,000keV.Other the potential radio isotope used comprises ¹¹c, ¹³n, ¹⁵o, ⁷⁵br, ¹⁹⁸au, ²²⁴ac, ¹²⁶i, ¹³³i, ⁷⁷br, ^113min, ⁹⁵ru, ⁹⁷ru, ¹⁰³ru, ¹⁰⁵ru, ¹⁰⁷hg, ²⁰³hg, ^121mte, ^122mte, ^125mte, ¹⁶⁵tm, ¹⁶⁷tm, ¹⁶⁸tm, ¹⁹⁷pt, ¹⁰⁹pd, ¹⁰⁵rh, ¹⁴²pr, ¹⁴³pr, ¹⁶¹tb, ¹⁶⁶ho, ¹⁹⁹au, ⁵⁷co, ⁵⁸co, ⁵¹cr, ⁵⁹fe, ⁷⁵se, ²⁰¹tl, ²²⁵ac, ⁷⁶br, ¹⁶⁹yb etc.Some useful diagnosis nucleic can comprise ¹⁸f, ⁵²fe, ⁶²cu, ⁶⁴cu, ⁶⁷cu, ⁶⁷ga, ⁶⁸ga, ⁸⁶y, ⁸⁹zr, ⁹⁴tc, ^94mtc, ^99mtc or ¹¹¹in.

Therapeutical agent can comprise photosensitizers or dyestuff.Fluorescent composition such as fluorescence dye and other chromophoric group or dyestuff (such as to visible responsive porphyrin) has been used to carry out test-and-treat pathology, namely with suitable rayed diseased region.In the treatment, this is called as optical radiation, phototherapy or photodynamic therapy.See the people such as Jori (volume), PHOTODYNAMICTHERAPYOFTUMORSANDOTHERDISEASES (LibreriaProgetto1985); VandenBergh, Chem.Britain (1986), 22:430.In addition, by monoclonal antibody and light-sensitive coloring agent coupling, for carrying out phototherapy.See people such as Mew, J.Immunol. (1983), 130:1473; Idem., CancerRes. (1985), 45:4380; The people such as Oseroff, Proc.Natl.Acad.Sci.USA (1986), 83:8744; Idem., Photochem.Photobiol. (1987), 46:83; The people such as Hasan, Prog.Clin.Biol.Res. (1989), 288:471; The people such as Tatsuta, LasersSurg.Med. (1989), 9:422; The people such as Pelegrin, Cancer (1991), 67:2529.

Other useful therapeutical agent can comprise oligonucleotide, the antisense oligonucleotide of particularly preferably lead antioncogene and oncoprotein such as bcl-2 or p53.The preferred form of therapeutic oligonucleotide is siRNA.

Diagnostic reagent

Diagnostic reagent is preferably selected from radionuclide, radiocontrast medium, paramagnetic ion, metal, fluorescent marker, chemiluminescent labels, acoustic contrast agent and photosensitive reagents.This type of diagnostic reagent is known and can uses this type of known diagnostic reagent any.The non-limiting example of diagnostic reagent can comprise radionuclide such as ¹¹⁰in, ¹¹¹in, ¹⁷⁷lu, ¹⁸f, 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, β or positron emitter.The paramagnetic ion used comprises chromium (III), manganese (II), iron (III), iron (III), cobalt (II), nickel (II), copper (II), rubidium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) or erbium (III).Acoustic contrast agent can comprise the liposome that liposomes is filled as gas.Radiopaque diagnostic reagent can be selected from compound, barium compound, gallium compound and thallium compound.Many kinds of fluorescent markers are known in the art, and include but not limited to fluorescein isothiocyanate, rhodamine, phycoerythrin, Phycocyanins, C-, allophycocyanin, Phthalyldicarboxaldehyde and fluorescamine.The chemiluminescent labels used can comprise luminol,3-aminophthalic acid cyclic hydrazide, different luminol,3-aminophthalic acid cyclic hydrazide, aromatic acridinium ester, imidazoles, acridinium salt or barkite.

The method of therapeutic treatment

Different embodiments relates to the method for the treatment experimenter such as cancer of Mammals (comprise people, raise and train or companion pets such as dog and cat), comprises to the dual specific immune cell factor DNL construct of described experimenter's administering therapeutic significant quantity.

In one embodiment, the Immunological diseases that DNL construct of the present invention can be utilized to treat can comprise such as joint disease such as ankylosing spondylitis, juvenile rheumatoid arthritis, rheumatoid arthritis; Sacred disease such as multiple sclerosis and myasthenia gravis; Pancreatic disease is diabetes such as, particularly juvenile onset diabetes; Gastrointestinal tract disease is chronic active hepatitis, celiac disease, ulcerative colitis, Crohn's disease, pernicious anemia such as; Skin disease is as psoriatic or scleroderma; Allergic disease such as asthma and transplanting associated conditions such as graft versus host disease (GVH disease) and allograft rejection.

Another kind of antibody (the another kind of antigen on described antibodies target cells or with described antigen-reactive) by simultaneously or one after the other administering therapeutic significant quantity carrys out using of supplementary dual specific immune cell factor DNL construct.Other MAb preferred comprises at least one humanization MAb, chimeric MAb or people MAb, it is selected from the MAb:CD4 with following substance reaction, 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, MUC5, Ia, MIF, HM1.24, HLA-DR, tenascin, Flt-3, VEGFR, PlGF, ILGF, IL-6, IL-25, tenascin, TRAIL-R1, TRAIL-R2, complement factor C5, oncoprotein or its combination.The different antibodies such as anti-CD19, anti-CD 20 and the anti-CD22 antibody that use are known to those skilled in the art.See, such as, the people such as Ghetie, CancerRes.48:2610 (1988); The people such as Hekman, CancerImmunol.Immunother.32:364 (1991); Longo, Curr.Opin.Oncol.8:353 (1996), the 5th, 798,554; 6,187,287; 6,306,393; 6,676,924; 7,109,304; 7,151,164; 7,230,084; 7,230,085; 7,238,785; 7,238,786; 7,282,567; 7,300,655; 7,312, No. 318 United States Patent (USP)s; With the 20080131363rd; 20080089838; 20070172920; 20060193865; 20060210475; 20080138333 and No. 20080146784 U.S. Patent Application Publication, are incorporated to by reference by the embodiment part of each section above-mentioned herein.

Also by simultaneously or one after the other use at least one therapeutical agent and supplement DNL construct therapy further.Such as " CVB " (1.5g/m ²endoxan, 200-400mg/m ²etoposide and 150-200mg/m ²carmustine) be the scheme being used for the treatment of non-He Jiejin lymphomas.The people such as Patti, Eur.J.Haematol.51:18 (1993).Other suitable associating chemotherapeutic treatment protocols is known to those skilled in the art.See, such as, the people such as Freedman, " Non-Hodgkin'slymphomas, " inCANCERMEDICINE, the 2nd volume, the 3rd edition, the people such as Holland (volume), 2028-2068 page (Lea & Febiger1993).As an example, the first-generation chemotherapeutic treatment protocols being used for the treatment of moderate non-He Jiejin lymphomas (NHL) comprises C-MOPP (endoxan, vincristine(VCR), Procarbazine and prednisone) and CHOP (endoxan, Dx, vincristine(VCR) and prednisone).Useful s-generation chemotherapeutic treatment protocols is m-BACOD (methotrexate, bleomycin, Dx, endoxan, vincristine(VCR), dexamethasone and folinic acid), and suitable third generation scheme is MACOP-B (methotrexate, Dx, endoxan, vincristine(VCR), prednisone, bleomycin and folinic acid).Other useful medicine comprises PB, bendamustine and Bryostatin-1.

DNL construct of the present invention can be prepared to prepare the composition of pharmaceutically useful, by by DNL construct and pharmaceutically suitable excipient composition in the mixture by currently known methods.Aseptic phosphate buffered saline buffer is an example of pharmaceutically suitable vehicle.Other suitable vehicle is known to those skilled in the art.See, such as, the people such as Ansel, PHARMACEUTICALDOSAGEFORMSANDDRUGDELIVERYSYSTEMS (pharmaceutical dosage form and drug delivery system), 5th edition (Lea & Febiger1990), and Gennaro (volume), REMINGTON ' SPHARMACEUTICALSCIENCES (Lei Mingdunshi pharmaceutical science), 18th edition (MackPublishingCompany1990), and revision version.

DNL construct preparation of the present invention can be used for intravenously to use, by such as bolus infusion or continuous infusion.Preferably, within the period being shorter than about 4 hours, infusion DNL construct in the period being more preferably shorter than about 3 hours.Such as, can in 30 minutes, preferably even 25-50mg before infusion in 15 minutes, infusion remainder in 2-3 subsequently hour.Injection preparation can be unit dosage, such as, be contained in ampoule, or to be contained in multi-dose container and to add sanitas.Composition can adopt the form of suspension, solution or emulsion such as in oiliness or aqueous vehicles, and can contain formula agent (formulatoryagents) such as suspension agent, stablizer and/or dispersion agent.Selectively, activeconstituents can be powder form, for using suitable vehicle (such as aseptic apirogen water) preparation before use.

Other pharmaceutical methods can be used for the acting duration of control DNL construct.Controlled release preparation realizes by using biocompatible polymer complexing or absorption DNL construct.Such as, biocompatible polymer comprises the polyanhydride copolymer matrix of ethylene-vinyl acetate copolymer matrix and stearic acid dimer and sebacic acid.The people such as Sherwood, Bio/Technology10:1446 (1992).From then on speed matrix discharged depends on the molecular weight of this DNL construct, the intramatrical amount of DNL construct and the size of dispersed particle.The people such as Saltzman, Biophys.J.55:163 (1989); The people such as Sherwood, the same.Other solid dosage is described in the people such as Ansel, PHARMACEUTICALDOSAGEFORMSANDDRUGDELIVERYSYSTEMS (pharmaceutical dosage form and drug delivery system), 5th edition (Lea & Febiger1990), with Gennaro (volume), REMINGTON ' SPHARMACEUTICALSCIENCES (Lei Mingdunshi pharmaceutical science), 18th edition (MackPublishingCompany1990), and revision version.

Also can by subcutaneous for DNL construct or be even applied to Mammals by other parental routes.In addition, use by continuous infusion or injected by single or multiple.Preferably, within the period being shorter than about 4 hours, more preferably infusion DNL construct within the period being shorter than about 3 hours.

More common, the application dosage for the DNL construct of people depends on the factors such as such as patient age, body weight, height, sex, general health and previous treatment history and changes.May need to provide the dosage of the DNL construct in about 1mg/kg to 25mg/kg scope as infusion in single dose intravenous to receptor, but also can according to circumstances need to use lower or higher dosage.The dosage used by 1-20mg/kg for 70kg patient is such as 70-1,400mg, or for the patient of 1.7 meters, dosage is 41-824mg/m ².This dosage can be repeated on demand, such as, once in a week, continue 4-10 week, or once in a week, continue 8 weeks or once in a week, continue 4 weeks.Administration frequencies that can also be lower, such as week about once, periods of months, or monthly or quarterly once, continue many months, as required in supportive care.

Selectively, with every 2 or 3 Monday a dosage use DNL construct, repeat at least 3 dosage altogether.Or construct described in administered twice weekly, continues 4-6 week.If dosage to be reduced to about 200-300mg/m ²(each dosage 340mg (patients for 1.7 meters) or 4.9mg/kg (patient for 70kg), can use weekly once or even secondary, continues for 4 to 10 weeks.Selectively, can dosage schedule be reduced, namely every 2 or 3 weeks once, continue 2-3 month.But, determine even to use higher dosage (such as weekly the or 20mg/kg of every 2-3 week) by intravenous infusion slowly, carried out the administration circulation of repetition.Optionally with other interval repeat administration scheme, and dosage can be provided by different parental routes (carrying out suitable adjustment to dosage and treatment plan).

In preferred embodiments, DNL construct is used for the treatment of cancer.The example of cancer includes but not limited to cancer, lymphoma, glioblastoma multiforme, melanoma, sarcoma and leukemia, myelomatosis or lymphoid malignancies (lymphoidmalignancies).Hereinafter point out the example more specifically of this type of cancer, comprise: squamous cell carcinoma (such as, epithelial squamous cell cancer), Ewing sarcoma, Wilms knurl, astrocytoma, lung cancer (comprises small cell lung cancer, nonsmall-cell lung cancer, the gland cancer of lung and the squamous cell carcinoma of lung), peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer (gastriccancer) or cancer of the stomach (comprising gastrointestinal cancer), carcinoma of the pancreas, glioblastoma multiforme, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, hepatocellular carcinoma, neuroendocrine tumour, medullary thyroid carcinoma (medullarythyroidcancer), the thyroid carcinoma of differentiation, mammary cancer, ovarian cancer, colorectal carcinoma, the rectum cancer, endometrial cancer or uterus carcinoma, salivary-gland carcinoma, kidney or kidney (renalcancer), prostate cancer, carcinoma vulvae, anus cancer, penile cancer and head and neck cancer.Term " cancer " comprises primary malignancy cell or tumour (such as, its cell does not also migrate to malignant cell or the tumour of the position of the health of the experimenter except the position of original malignant tumour or tumour) and Secondary cases malignant cell or tumour (such as, by malignant cell or tumour cell to the Secondary cases position different from the position of primary tumor transfer, move the malignant cell or tumour that produce).The cancer being suitable for methods for the treatment of of the present invention comprises the cell of expression, process LAN or unconventionality expression IGF-1R.

Other example of cancer or malignant tumour includes but not limited to: children acute lymphoblastic leukaemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia (AcuteLymphocyticLeukemia), acute myeloid leukemia, adrenocortical carcinoma, adult's (primary) hepatocellular carcinoma, adult's (primary) liver cancer, adult acute's Lymphocytic leukemia, adult acute's myeloid leukemia, adult He Jiejin lymphomas, adult lymphoid chronic myeloid leukemia, adult non-He Jiejin lymphomas, adult primary liver cancer, adult soft tissue sarcoma, AIDS-associated lymphoma, AIDS-associated malignancies, the rectum cancer, astrocytoma, cholangiocarcinoma, bladder cancer, osteocarcinoma, brain stem glioma, brain tumor, mammary cancer, renal plevis and ureteral cancer, primary central nervous system lymphoma, central nervous system lymphoma, cerebellar astrocytoma (CerebellarAstrocytoma), cerebral astrocytoma (CerebralAstrocytoma), cervical cancer, children's (primary) hepatocellular carcinoma, children's (primary) liver cancer, children acute Iymphoblastic leukemia (ChildhoodAcuteLymphoblasticLeukemia), children acute myeloid leukemia, children's brain stem glioma, Cerebellar Astrocytoma in Children. An (ChildhoodCerebellarAstrocytoma), Cerebellar Astrocytoma in Children. An ChildhoodCerebralAstrocytoma), children's extracranial germ cell tumour (ChildhoodExtracranialGermCellTumors), children's Hokdkin disease (ChildhoodHodgkin'sDisease), children He Jiejin lymphomas, children's hypothalamus and visual pathway neurospongioma, children's Lymphocytic leukemia (ChildhoodLymphoblasticLeukemia), Children Medulloblastoma (ChildhoodMedulloblastoma), children non-He Jiejin lymphomas, the outer embryoma of original nerve on children's pineal gland and curtain, childhood idiopathic liver cancer, Children Rhabdomyosarcoma, children soft tissue sarcoma, children's visual pathway and hypothalamus neurospongioma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectal carcinoma, cutaneous T cell lymphoma, endocrine islet cell carcinoma, endometrial cancer, ependymoma, epithelial cancer, the esophageal carcinoma, Ewing sarcoma and related neoplasms, exocrine pancreas cancer, the outer blastoma (ExtracranialGermCellTumor) of cranium, the outer blastoma (ExtragonadalGermCellTumor) of sexual gland, cholangiocarcinoma, cancer eye, female mammary gland cancer, familial splenic anemia (Gaucher'sDisease), carcinoma of gallbladder, cancer of the stomach, gastrointestinal associated cancers (GastrointestinalCarcinoidTumor), stomach and intestine knurl (GastrointestinalTumors), gonioma, gestational trophoblastic tumor, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, He Jiejin lymphomas, hypergammaglobulinemia (Hypergammaglobulinemia), hypopharyngeal cancer, intestinal cancer, intraocular melanoma, islet-cell carcinoma, islet cells carcinoma of the pancreas, Kaposi sarcoma, kidney, laryngocarcinoma, lip and oral carcinoma, liver cancer, lung cancer, lymphoproliferative disorders (LymphoproliferativeDisorders), macroglobulinemia, male mammary cancer, malignant mesothe (MalignantMesothelioma), malignant thymoma, medulloblastoma, melanoma, mesothelioma, transitivity primary squamous neck cancer (MetastaticOccultPrimarySquamousNeckCancer), transitivity primary squamous neck cancer (MetastaticPrimarySquamousNeckCancer), transitivity squamous neck cancer (MetastaticSquamousNeckCancer), multiple myeloma, multiple myeloma/plasma cell tumor, myelodysplastic syndrome, myelogenous leukemia (MyelogenousLeukemia), myeloid leukemia (MyeloidLeukemia), myeloproliferative disease, nose and paranasal sinuses cancer (NasalCavityandParanasalSinusCancer), nasopharyngeal carcinoma, neuroblastoma, non-He Jiejin lymphomas, non-melanoma skin carcinoma, nonsmall-cell lung cancer, invisible primary transitivity squamous neck cancer (OccultPrimaryMetastaticSquamousNeckCancer), oropharynx cancer, osteosarcoma/malignant fibrous sarcoma, osteosarcoma/malignant fibrous histiocytoma, osteosarcoma/malignant fibrous histiocytoma of bone, epithelial ovarian cancer, ovarian germ cell tumors, ovary low potential malignancy potential tumour (OvarianLowMalignantPotentialTumor), carcinoma of the pancreas, paraproteinemia (Paraproteinemias), polycythemia vera, parathyroid carcinoma, penile cancer, pheochromocytoma, pituitary tumor, primary central nervous system lymphoma (PrimaryCentralNervousSystemLymphoma), primary hepatocarcinoma, prostate cancer, the rectum cancer, renal cell carcinoma, renal plevis and carcinoma of ureter (RenalPelvisandUreterCancer), retinoblastoma, rhabdosarcoma, salivary gland carcinoma, sarcoidosis sarcoma, Richter scale syndrome (SezarySyndrome) is pricked in match, skin carcinoma, small cell lung cancer, carcinoma of small intestine, soft tissue sarcoma, squamous neck cancer (SquamousNeckCancer), cancer of the stomach, primitive neuroectodermal and pinealoma on curtain, t cell lymphoma, carcinoma of testis, thymoma, thyroid carcinoma, renal plevis and transitional cell carcinoma of ureter (TransitionalCellCanceroftheRenalPelvisandUreter), renal plevis and transitional cell carcinoma of ureter (TransitionalRenalPelvisandUreterCancer), trophoblastic tumor (TrophoblasticTumors), ureter and renal plevis cell carcinoma (UreterandRenalPelvisCellCancer), urethral carcinoma, uterus carcinoma, sarcoma of uterus, carcinoma of vagina, visual pathway and hypothalamus neurospongioma, carcinoma vulvae, Walden Si Telun macroglobulinemia, the nephroblastoma and other excess proliferative disease any, except true tumor is formed, be all arranged in tract listed above.

Described herein and claimed method and composition can be used for treating pernicious or state and for stoping the progress to tumorigenesis state (neoplasticstate) or malignant state (including but not limited to those obstacles above-mentioned) before worsening.This type of purposes be applicable to known or suspect proceed to tumorigenesis or cancer, particularly wherein by hyperplasia, change disease that raw (metaplasia) or dysplasia forms the most especially non-tumorigenesis Growth of Cells occurred (about the summary of this type of misgrowth condition, see Robbins and Angell, BasicPathology, 2nd edition, W.B.SaundersCo., Philadelphia, pp.68-79 (1976)) the state of progress.

The tendency of dysplasia normally cancer, and be mainly seen in epithelium.It is the most disorderly form of non-tumorigenesis Growth of Cells, comprises the loss of structure towards (architecturalorientation) of individual cells consistence and cell.When there is chronic stimulation or inflammation, dysplasia occurs characteristically.The dysplasia obstacle that can be treated includes but not limited to anhidrotic ectodermal dysplasia (anhidroticectodermaldysplasia), heteropleural dysplasia (anterofacialdysplasia), asphyxiating thoracic dysplasia, atriodigital dysplasia (atriodigitaldysplasia), broncho-pulmonary dysplasia, cerebral dysplasia (cerebraldysplasia), cervical dysplasias is bad, chondroectodermal dysplasia, CCD, congenital ectodermal dysplasia, craniodiaphyseal dysplasia (craniodiaphysialdysplasia), cranium wrist metatarsal underdevelopment (craniocarpotarsaldysplasia), skull metaphysis underdevelopment (craniometaphysialdysplasia), dentinal dysplasia (dentindysplasia), camurati-Engelmann disease (diaphysialdysplasia), ectodermal dysplasia (ectodermaldysplasia), enamel hypoplasia (enameldysplasia), encephalo-ophthalmic dysplasia (encephalo-ophthalmicdysplasia), dysplasia epiphysialis hemimelia (dysplasiaepiphysialishemimelia), multiple epiphyseal dysplasia (dysplasiaepiphysialismultiplex), chondrodystrophia congenita punctata (dysplasiaepiphysialispunctata), epithelial dysplasia (epithelialdysplasia), face-refer to (toe)-genital development bad (faciodigitogenitaldysplasia), familial fibrous dysplasia of jaw (familialfibrousdysplasiaofjaws), familial white pleat heteroplasia (familialwhitefoldeddysplasia), fibromuscular dysplasia (fibromusculardysplasia), fibrous dysplasia (fibrousdysplasiaofbone), florid osseous dysplasia (floridosseousdysplasia), heredity kidney-retinal dysplasia (hereditaryrenal-retinaldysplasia), perspiration is ectodermal dysplasia (hidroticectodermaldysplasia), hypohidrotic ectodermal dysplasia (hypohidroticectodermaldysplasia), lymphopenia thymic aplasia (lymphopenicthymicdysplasia), breast dysplasia (mammarydysplasia), lower maxillofacial development bad (mandibulofacialdysplasia), metaphysis dysplasia (metaphysialdysplasia), cover end Buddhist nun heteroplasia (Mondinidysplasia), single fibrous dysplasia of bone (monostoticfibrousdysplasia), mucous epithelium dysplasia (mucoepithelialdysplasia), multiple epiphyseal dysplasia (multipleepiphysialdysplasia), eye-Er-spinal syndromes (oculoauriculovertebraldysplasia), oculo-dental-digital (toe) dysplasia (oculodentodigitaldysplasia), oculovertebral dysplasia (oculovertebraldysplasia), odontodysplasia (odontogenicdysplasia), opthalmomandibulomelicdysplasia, periapical dental cement dysplasia (periapicalcementaldysplasia), polyostotic fibrous dysplasia (polyostoticfibrousdysplasia), pseudoachondroplasia spondylo epiphyseal dysplasia (pseudoachondroplasticspondyloepiphysialdysplasia), retinal dysplasia (retinaldysplasia), every-hypoplasia of optic nerve (septo-opticdysplasia), vertebra aplasia of epiphysis (spondyloepiphysialdysplasia) and ventriculoradialdysplasia.

Before the other tumour that can be treated, obstacle includes but not limited to optimum hyperplasia sexual dysfunction (such as, innocent tumour, fibrocystic conditions, tissue hyperplasia, polyp intestinal or adenoma and esophagus dysplasia), hickie, keratosis, bowen's disease, farmer's skin disease (Farmer'sSkin), solar cheilitis and solar keratosis.

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

Other excess proliferative disease, obstacle and/or state include but not limited to progress and/or the transfer of malignant tumour and associated disorders, and described malignant tumour and associated disorders are that such as leukemia (comprises acute leukemia (such as, acute lymphoblastic leukemia, acute myelocytic leukemia (comprises myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemias, monocytic leukemia and erythroleukemia)) and chronic leukemia (such as, chronic myelocytic (granulocyte) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (such as, Hokdkin disease and non Hodgkin's disaese), multiple myeloma, Walden Si Telun macroglobulinemia, heavy chain disease and solid tumor, include but not limited to sarcoma and cancer such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, Ewing sarcoma, leiomyosarcoma, rhabdosarcoma, colorectal carcinoma, carcinoma of the pancreas, mammary cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, rodent cancer, gland cancer, syringocarcinoma, sebaceous carcinoma, papillary carcinoma, papillary carcinoma, cystadenocarcinoma, medullary carcinoma, bronchiogenic cancer, renal cell carcinoma, hepatoma (hepatoma), cholangiocarcinoma, choriocarcinoma, spermocytoma, embryonal carcinoma, the nephroblastoma ((Wilm'stumor), cervical cancer, tumor of testis, lung cancer, small cell lung cancer, bladder cancer, cell carcinoma, neurospongioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma (emangioblastoma), acoustic tumor, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma.

Expression vector

Other embodiment can relate to the DNA sequence dna of the nucleic acid of the antibody, antibody fragment, cytokine or the composition fusion rotein that comprise encoding D NL construct.Fusion rotein can comprise the antibody or fragment or cytokine that are connected to such as AD or DDD part.

Various embodiment relates to the expression vector comprising DNA sequences encoding.Described carrier can comprise the light chain of encoding human immunoglobulin and the sequence of CH and hinge area, this sequence can be connected with chimeric, humanization or people's variable region sequences.Described carrier can be included in the promotor of the protein of expressing coding in selected host cell, enhanser and signal sequence or leader sequence extraly.Useful especially carrier is pdHL2 or GS.More preferably, light chain and CH and hinge area can from people EU myelomatosis immunoglobulin (Ig)s, wherein optionally allotope is set up amino acid whose at least one change over the amino acid found in different I gG1 allotype, and wherein optionally can substitute the amino acid 253 of the heavy chain of the EU based on EU numbering system with L-Ala.See people such as Edelman, Proc.Natl.Acad.SciUSA63:78-85 (1969).In other embodiments, IgG1 sequence transitions can be become IgG4 sequence.

It will be understood by those skilled in the art that genetic engineering modified expression vector and Insertion Into Host Cell are well known in the art with the method expressing engineered protein and only normal experiment.Describe in the such as the 7th, 531, No. 327 and the 7th, 537, No. 930 United States Patent (USP)s (its embodiment part be incorporated to by reference herein) separately for the antibody of cloning by expression or the host cell of fragment and method.

Test kit

Various embodiment can relate to the test kit containing the component being suitable for the illing tissue treating or diagnose patient.Exemplary kit can comprise one or more DNL constructs described herein.If comprise the composition using component not make and sent by digestive tube (as passed through oral delivery), then also can comprise can by the equipment of some other approach delivery of agents box components for test kit.Be a syringe for using the device type of such as potential delivery, it is for being expelled to described composition in subject.Also inhalation device can be used.In certain embodiments, therapeutical agent can be provided with the form of prefilled syringe or automatic injection pen (autoinjectionpen) that sterile liquid formulations or freeze-dried preparation are housed.

Described reagent constituents can packaging together or separately be packaged in two or more containers.In certain embodiments, described container can be the phial comprising aseptic, the freeze-dried preparation being applicable to the composition rebuilding (reconstitution).Test kit also can comprise one or more damping fluids being applicable to rebuild and/or dilute other reagent.Other spendable container includes but not limited to: bag, dish, box, pipe etc.Reagent constituents can also be preserved in the above-described container in sterile packed.Another integral part that can comprise is to the operation instruction of the personnel using test kit.

Embodiment

The following example is provided to illustrate, but does not limit claim of the present invention.

Embodiment 1. is for generation of the general strategy of module Fab subunit

Fab module can be prepared into the fusion rotein comprising DDD or AD sequence.For each Fab fusion rotein develops independently transgenic cell line.When preparing, if needed, module can be carried out purifying or be kept in the supernatant liquor of cell culture.After preparation, any DDD ₂can arbitrary combination between module and any A/D module, to produce trivalent DNL construct.

Plasmid vector pdHL2 is for generation of Multiple Antibodies and the construct based on antibody.See people such as Gillies, JImmunolMethods (1989), 125:191-202; The people such as Losman, Cancer (Phila) (1997), 80:2660-6.The synthesis of bicistronic mRNA mammalian expression vector mediation IgG heavy chain and light chain.The carrier sequence overwhelming majority of many different I gG-pdHL2 constructs is all identical, only there are differences in variable region (VH and VL) sequence.Use biology tool well known by persons skilled in the art, these IgG expression vectors can be converted into Fab-DDD or Fab-AD expression vector.In order to produce Fab-DDD expression vector, the hinge area of heavy chain, the encoding sequence of CH2 and CH3 structural domain replace with the sequence (being called DDD1) of coding front 4 residues of described hinge, the Gly-Ser linker of 14 residues and front 44 residues of people RII α.In order to produce Fab-AD expression vector, the hinge area of IgG, the sequence of CH2 and CH3 structural domain replace with the sequence (being called AD1) of the synthesis AD of coding front 4 residues of described hinge, the Gly-Ser linker of 15 residues and 17 residues of AKAP-IS by name, this sequence system uses information biology and peptide array technique to produce, and the avidity (0.4nM) be combined with RII α dimer that display is very high.See the people Proc.Natl.Acad.Sci. such as Alto, U.S.A (2003), 100:4445-50.

Devise two shuttle vectorss, being beneficial to IgG-pdHL2 vector is Fab-DDD1 or Fab-AD1 expression vector, as described below.

The preparation of CH1

CH1 structural domain system uses pdHL2 plasmid vector as template, is obtained by pcr amplification.Left side PCR primer is made up of the upstream (5 ') of CH1 structural domain and SacII restriction endonuclease site (i.e. CH1 encoding sequence 5 ').Right primer is made up of front 4 residues of hinge of encoding and the sequence of short linker (latter two codon comprises BamHI restriction enzyme site).

5 ' of CH1 Left primer

5’GAACCTCGCGGACAGTTAAG-3’(SEQIDNO:63)

cH1+G ₄ on the right side of S-Bam

5’GGATCCTCCGCCGCCGCAGCTCTTAGGTTTCTTGTCCACCTTGGTGTTGCTGG-3’(SEQIDNO:64)

410bpPCR amplimer is cloned into pGemTPCR cloning vector (Promega, Inc.), and then screening and cloning is to obtain the inset in T7 (5 ') direction.

(G ₄s) ₂the structure of DDD1

SigmaGenosys (Haverhill, UK) is used to synthesize duplex oligonucleotide (called after (G ₄s) ₂dDD1) with the aminoacid sequence of encoding D DD1, have 11 residues of connection peptides before described DDD1, wherein the first two codon comprises BamHI restriction enzyme site.Termination codon gives and is positioned at 3 ' end with EagI restriction enzyme site.The peptide sequence of coding is as follows.

GSGGGGSGGGG SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQIDNO:65)

Synthesize two oligonucleotide (on called after RIIA1-44 and under RIIA1-44, their 3 ' end has the overlap of 30 base pairs) (SigmaGenosys), and they are combined 154 base pairs placed in the middle comprising 174bpDDD1 sequence.Oligonucleotide is annealed, then uses Taq polysaccharase to carry out primer extension reaction.

on RIIA1-44

5’GTGGCGGGTCTGGCGGAGGTGGCAGCCACATCCAGATCCCGCCGGGGCTCACGGAGCTGCTGCAGGGCTACACGGTGGAGGTGCTGCGACAG-3’(SEQIDNO:66)

under RIIA1-44

5’GCGCGAGCTTCTCTCAGGCGGGTGAAGTACTCCACTGCGAATTCGACGAGGTCAGGCGGCTGCTGTCGCAGCACCTCCACCGTGTAGCCCTG-3’(SEQIDNO:67)

After primer extension, duplex described in following primer pair is used to increase:

g4SBam-is left

5’-GGATCCGGAGGTGGCGGGTCTGGCGGAGGT-3’(SEQIDNO:68)

it is right that 1-44 stops Eag

5’-CGGCCGTCAAGCGCGAGCTTCTCTCAGGCG-3’(SEQIDNO:69)

This amplimer is cloned into pGemT, then screens the inset in T7 (5 ') direction.

(G ₄s) ₂the structure of-AD1

Synthesis duplex oligonucleotide (called after (G ₄s) ₂-AD1) (SigmaGenosys), with the aminoacid sequence of the AD1 that encodes, have 11 residues of connection peptides before described AD1, wherein the first two codon comprises BamHI restriction enzyme site.Terminator codon and EagI restriction enzyme site are positioned at 3 ' end.The peptide sequence of coding is as follows.

GSGGGGSGGGGS QIEYLAKQIVDNAIQQA(SEQIDNO:70)

Synthesize two complementations and have overlapping oligonucleotide (on called after AKAP-IS and under AKAP-IS).

on AKAP-IS

5’GGATCCGGAGGTGGCGGGTCTGGCGGAGGTGGCAGCCAGATCGAGTACCTGGCCAAGCAGATCGTGGACAACGCCATCCAGCAGGCCTGACGGCCG-3’(SEQIDNO:71)

under AKAP-IS

5’CGGCCGTCAGGCCTGCTGGATGGCGTTGTCCACGATCTGCTTGGCCAGGTACTCGATCTGGCTGCCACCTCCGCCAGACCCGCCACCTCCGGATCC-3’(SEQIDNO:72)

Duplex described in following primer pair is used to carry out pcr amplification:

g4SBam-is left

5’-GGATCCGGAGGTGGCGGGTCTGGCGGAGGT-3’(SEQIDNO:73)

it is right that AKAP-IS stops Eag

5’-CGGCCGTCAGGCCTGCTGGATG-3’(SEQIDNO:74)

This amplimer is cloned into pGemT carrier, then screens the inset in T7 (5 ') direction.

Connect DDD1 and CH1

Use BamHI and NotI restriction enzyme the 190bp fragment of encoding D DD1 sequence to be cut from pGemT, then this fragment is connected to the same loci of CH1-pGemT, to produce shuttle vectors CH1-DDD1-pGemT.

Connect AD1 and CH1

Use BamHI and NotI the 110bp fragment containing AD1 sequence to be cut from pGemT, then this fragment is connected to the same loci of CH1-pGemT, to produce shuttle vectors CH1-AD1-pGemT.

CH1-DDD1 or CH1-AD1 is cloned into the carrier based on pdHL2

Use this modular design, CH1-DDD1 or CH1-AD1 all can be contained in any IgG construct in pdHL2 carrier.By removing the SacII/EagI restriction fragment (CH1-CH3) of pdHL2, and being replaced with the SacII/EagI fragment of CH1-DDD1 or CH1-AD1 cut from corresponding pGemT shuttle vectors, namely whole CH replaces with one of above-mentioned construct.

N-end DDD structural domain

The position of DDD or AD is not limited to the C-terminal of CH1.Transform wherein DDD1 sequence and be connected to the aminoterminal construct of VH structural domain.

Embodiment 2: expression vector

The structure of h679-Fd-AD1-pdHL2

H679-Fd-AD1-pdHL2 is the expression vector for generation of h679Fab, and wherein AD1 is coupled to the C-terminal of the CH1 structural domain of Fd by the flexible Gly/Ser peptide transcribed spacer be made up of 14 amino-acid residues.The SacII/EagI fragment comprising the carrier based on pdHL2 of the variable region of h679 is replaced with CH1-AD1 fragment (being cut from CH1-AD1-SV3 shuttle vectors by SacII and EagI), and namely this carrier is converted into h679-Fd-AD1-pdHL2.

The structure of C-DDD1-Fd-hMN-14-pdHL2

C-DDD1-Fd-hMN-14-pdHL2 is the expression vector for generation of fusion rotein C-DDD1-Fab-hMN-14, and in described fusion rotein, DDD1 is connected to the CH1 C-terminal of hMN-14Fab by flexible peptide spacer.Plasmid vector hMN14 (I)-pdHL2 (for generation of hMN-14IgG) is by digesting by SacII and EagI restriction endonuclease to remove CH1-CH3 structural domain, then insert CH1-DDD1 fragment (being cut from CH1-DDD1-SV3 shuttle vectors by SacII and EagI), be namely converted into C-DDD1-Fd-hMN-14-pdHL2.

The structure of N-DDD1-Fd-hMN-14-pdHL2

N-DDD1-Fd-hMN-14-pdHL2 be for generation of comprise two copy fusion rotein N-DDD1-Fab-hMN-14 stablize dimeric expression vector, in described fusion rotein, DDD1 is connected to the N-terminal of the VH of hMN-14Fab by flexible peptide spacer.As follows described expression vector is transformed.Two primer pair DDD1 structural domains shown in below use carry out pcr amplification.

dDD1Nco is left

5’CCATGGGCAGCCACATCCAGATCCCGCC–3’(SEQIDNO:75)

dDD1-G ₄ sBam is right

5’GGATCCGCCACCTCCAGATCCTCCGCCGCCAGCGCGAGCTTCTCTCAGGCGGGTG-3’(SEQIDNO:76)

As the result of PCR, namely the encoding sequence comprising the part of BamHI restriction enzyme site in NcoI restriction enzyme site and linker is connected to 5 ' and 3 ' end respectively.170bpPCR amplimer is cloned into pGemT carrier, and then screening and cloning is to obtain the inset in T7 (5 ') direction.194bp inset is cut from described pGemT carrier by NcoI and SalI restriction enzyme, and is cloned into described SV3 shuttle vectors (using identical enzymic digestion to be prepared from), to generate intermediate carrier DDD1-SV3.

Oligonucleolide primers shown in below use carries out pcr amplification to hMN-14Fd sequence.

the left G4SBam of hMN-14VH

5’-GGATCCGGCGGAGGTGGCTCTGAGGTCCAACTGGTGGAGAGCGG-3’(SEQIDNO:77)

cH1-C stops Eag

5’-CGGCCGTCAGCAGCTCTTAGGTTTCTTGTC–3’(SEQIDNO:78)

As the result of PCR, namely the encoding sequence of the part of BamHI restriction enzyme site and linker is connected to 5 ' end of amplimer.Terminator codon and EagI restriction enzyme site are then connected to 3 ' end.This 1043bp amplimer is cloned into pGemT.Use BamHI and EagI restriction enzyme to be cut from pGemT by hMN-14-Fd inset, then this inset is connected to DDD1-SV3 carrier (using identical enzymic digestion to be prepared from), to produce construct N-DDD1-hMN-14Fd-SV3.

Use XhoI and EagI restriction enzyme N-DDD1-hMN-14Fd sequence to be cut, then this 1.28kb Insert Fragment is connected to the carrier segments prepared through same enzyme digestion C-hMN-14-pdHL2.Final expression vector is N-DDD1-Fd-hMN-14-pDHL2.

The generation of embodiment 3.h679-Fab-AD1 and purifying

679 antibodies HSG target antigens and affinity chromatography can be utilized to carry out purifying.H679-Fd-ADl-pdHL2 carrier Sal1 restriction endonuclease digestion and linearized, then by electroporation method transfection Sp/EEE myeloma cell.The synthesis of this two-cistron expression vector mediation h679 κ light chain and h679Fd-AD1 and secretion, they combine and form h679Fab-AD1.After electroporation, be coated with 96 hole tissue culturing plates with described cell, and select Transfected clones with 0.05 μM of methotrexate (MTX).Use the protein expression of microtiter plate by ELISA screening and cloning of coating BSA-IMP-260 (HSG) conjugate, and use the Goat anti human Fab puting together HRP to detect.Analyze using the BIAcore of HSG (IMP-239) sensor chip and be used for determining productive rate by the initial slope measured available from the diluted medium sample of injection.The clone that output is the highest has the initial productive rate being about 30mg/L.Use single step IMP-291 affinity chromatography, be purified into from 4.5 liters of spinner culture things and amount to 230mgh679-Fab-AD1.Before adding to IMP-291-affigel post, substratum hyperfiltration process concentrates about 10 times.Use PBS that this post is eluted to baseline, then use 1M imidazoles, 1mMEDTA, 0.1MNaAc, pH4.5 wash-out h679-Fab-AD1.The SE-HPLC of elutriant is analyzed and shows to there is the single sharp peak of retention time (9.63 minutes) corresponding to 50kDa albumen (not shown).In reduction SDS-PAGE analyzes, only there are two of the polypeptide fraction representing h679-AD1 visible (not shown)s of band.

The generation of embodiment 4.N-DDD1-Fab-hMN-14 and C-DDD1-Fab-hMN-14 and purifying

C-DDD1-Fd-hMN-14-pdHL2 and N-DDD1-Fd-hMN-14-pdHL2 carrier enters the derivative myeloma cell of Sp2/0 by electroporation transfection.C-DDD1-Fd-hMN-14-pdHL2 is two-cistron expression vector, and the synthesis of its mediation hMN-14 κ light chain and hMN-14Fd-DDD1 and secretion, they are combined to form C-DDD1-hMN-14Fab.N-DDD1-hMN-14-pdHL2 is two-cistron expression vector, and the synthesis of its mediation hMN-14 κ light chain and N-DDD1-Fd-hMN-14 and secretion, they are combined to form N-DDD1-Fab-hMN-14.Each fusion rotein all forms stable homodimer by the interaction of DDD1 structural domain.

After electroporation, be coated with 96 hole tissue culturing plates with described cell, and select Transfected clones with 0.05 μM of methotrexate (MTX).Use the protein expression of microtiter plate by ELISA screening and cloning of coating WI2 (the large mouse-anti id monoclonal antibody for hMN-14), and use the Goat anti human Fab puting together HRP to detect.The initial productive rate of C-DDD1-Fab-hMN14Fab and the N-DDD1-Fab-hMN14Fab clone that output is the highest is respectively 60mg/L and 6mg/L.

Use AD1-Affigel affinity purification N-DDD1-hMN-14 and C-DDD1-hMN-14

Use DDD/AD to interact and affinity purification is carried out to the construct comprising DDD1.AD1-C is peptide be made up of ADI sequence and C-terminal cysteine residues by synthesis preparation, and described cysteine residues is for being coupled to Affigel (reaction according to sulfydryl and sym-dichloroacetic anhydride) by this peptide.Comprise a of DDD ₂structure is specific binding AD1-C-Affigel resin under neutral ph, and can under low pH (such as, pH2.5) wash-out.

Use single step AD1-C affinity chromatography, be purified into from 1.2 liters of spinner culture things and amount to 81mgC-DDD1-Fab-hMN-14.Before adding to AD1-C-affigel post, substratum hyperfiltration process concentrates about 10 times.Use PBS that this post is eluted to baseline, then use 0.1M glycine, pH2.5 wash-out C-DDD1-Fab-hMN-14.The SE-HPLC of elutriant is analyzed and shows to there is retention time (8.7 minutes) the single protein peak consistent with 107kDa albumen (not shown).Purity is also through reduction SDS-PAGE analysis confirmation, and only display is contemplated to two band (not shown)s of the molecular size of two polypeptide fraction of C-DDD1-Fab-hMN-14.

As mentioned above, use single step ADI-C affinity chromatography, be purified into from 1.2 liters of spinner culture things and amount to 10mgN-DDD1-hMN-14.The SE-HPLC of elutriant is analyzed and shows to exist the retention time (8.77 minutes) the single protein peak consistent with 107kDa albumen (not shown) that are similar to C-DDD1-Fab-hMN-14.Reduction SDS-PAGE analyzes only display and corresponds to two band (not shown)s of the polypeptide fraction of N-DDD1-Fab-hMN-14.

The binding activities of C-DDD1-Fab-hMN-14 is analyzed by the SE-HPLC of sample and is determined, in described sample, this trial target mixes from the WI2 of different amount.WI2Fab and C-DDD1-Fab-hMN-14 shows three peaks by the sample that the mixed in molar ratio of 0.75:1 is made, corresponding to unconjugated C-DDD1-Fab-hMN14 (8.71 minutes), the C-DDD1-Fab-hMN-14 (7.95 minutes) in conjunction with a WI2Fab and C-DDD1-Fab-hMN14 (7.37 minutes) (not shown) in conjunction with two WI2Fab.When the mol ratio of WI2Fab and C-DDD1-Fab-hMN-14 that the sample analyzed comprises is 4, only observe the unimodal (not shown) of 7.36 minutes.These results prove, hMN14-Fab-DDD1 is dimer, and have two active binding site.Use N-DDD1-Fab-hMN-14 to repeat this experiment, the result obtained is closely similar.

Competitive ELISA proves, C-DDD1-Fab-hMN-14 with N-DDD1-Fab-hMN-14 is all combined with CEA with the avidity similar with hMN-14IgG, and avidity is significantly better than unit price hMN-14Fab (not shown).Use the fusion rotein coating ELISA comprising the special CEA epi-position (A3B3) of hMN-14 dull and stereotyped.

Embodiment 5.a ₂the formation of b mixture

A ₂the evidence that b complex body is formed is by comprising equimolar C-DDD1-Fab-hMN-14 (as a ₂) and h679-Fab-AD1 (SE-HPLC as mixture b) analyzes and first provides.When this sample is analyzed, observe the simple spike that retention time is 8.40 minutes, this is greater than the consistent (not shown) of new albumen of any independent h679-Fab-AD1 (9.55 minutes) or C-DDD1-Fab-hMN-14 (8.73 minutes) with defining.As hMN-14F (ab ') ₂mix with h679-Fab-AD1, or during C-DDD1-Fab-hMN-14 and 679-Fab-NEM mixing, all do not observe this upfield shift, prove to interact clearly by DDD1 and AD1 domain mediates.H679-Fab-AD1 and N-DDD1-Fab-hMN-14 is used to obtain closely similar result (not shown).

The specificity using BIAcore to prove further and identify between DD1 and AD1 fusion rotein interacts.This experimentation is as follows: the surface first h679-Fab-AD1 or 679-Fab-NEM being attached to (IMP239) sensor chip of highdensity HSG coupling, then injects C-DDD1-Fab-hMN-14 or hMN-14F (ab ') ₂test.As expected, when injecting the latter, h679-Fab-AD1 and C-DDD1-Fab-hMN-14 is only had to combine the further increase (not shown) causing response unit.H679-Fab-AD1 and C-DDD1-Fab-hMN-14 is used to obtain similar result (not shown).

Carry out balancing SE-HPLC experiment to measure the interactional binding affinity of specificity being present in each fusion rotein between AD1 and DDD1.Find the dissociation constant (K of the combination of the commercial sample of h679-Fab-AD1 and C-DDD1-Fab-hMN-14, N-DDD1-hMN-14 and recombinant human RII α _d) be respectively 15nM, 8nM and 30nM.

The affinity purification of embodiment 6.DDD or AD fusion rotein

General affinity chromatography system is developed by producing DDD or the AD albumen with more low-affinity stop.The DDD formed by RI α dimer with compared with RII α 1/500 avidity (225nM) in conjunction with AKAP-IS (AD1).Therefore, the RI α dimer of 44 amino-acid residue formation in the past can be produced and be coupled to resin to produce the affinity matrix being used for any fusion rotein containing AD1 of purifying.

There is many comparatively low-affinities (0.1 μM) AKAP anchoring domain in nature.If needed, the amino acid replacement of very predictable can be introduced to reduce described binding affinity further.Low-affinity AD by synthesis or biological method preparation, and can be coupled to the affinity purification of resin for any DDD1 fusion rotein.

Embodiment 7. is for generation of the carrier of disulfide linkage rock steady structure

N-DDD2-Fd-hMN-14-pdHL2

N-DDD2-hMN-14-pdHL2 is the expression vector for generation of N-DDD2-Fab-hMN-14, and it has dimerization and the dockerin domain sequence of the DDD2 be connected with the N-terminal of Fd.DDD2 is coupled to V by the Gly/Ser peptide linker of 15 amino-acid residues _hstructural domain.DDD2 has the dimerization sequence identical with the sequence of DDD1 and docking sequence, but has cysteine residues before them.

As follows described expression vector is transformed.The complementary oligonucleotide (DDD2 is upper with under DDD2) of the overlap of the 1-13 position residue of DDD2 is comprised by synthesis preparation two.Described oligonucleotide is annealed, and by T4 polynucleotide kinase (PNK) phosphorylation, to form applicable and overhanging of being connected with the DNA that restriction endonuclease NcoI and PstI digests respectively 5 ' with 3 ' end.

on DDD2

5’CATGTGCGGCCACATCCAGATCCCGCCGGGGCTCACGGAGCTGCTGCA-3’(SEQIDNO:79)

under DDD2

5’GCAGCTCCGTGAGCCCCGGCGGGATCTGGATGTGGCCGCA-3’(SEQIDNO:80)

Described duplex DNA is connected with carrier segments DDD1-hMN14Fd-SV3 (preparing by digesting with NcoI and PstI), to generate middle construct DDD2-hMN14Fd-SV3.Use XhoI and EagI restriction endonuclease, the 1.28kb Insert Fragment comprising DDD2-hMN14Fd encoding sequence is cut from described middle construct, then this fragment is connected to the hMN14-pdHL2 carrier DNA using same enzyme digestion preparation.Final expression vector is N-DDD2-Fd-hMN-14-pdHL2.

C-DDD2-Fd-hMN-14-pdHL2

C-DDD2-Fd-hMN-14-pdHL2 is the expression vector for generation of C-DDD2-Fab-hMN-14, and it has the dimerization and the dockerin domain sequence that are connected to the DDD2 of the C-terminal of Fd by the Gly/Ser peptide linker of 14 amino-acid residues.As follows described expression vector is transformed.The complementary oligonucleotide of the overlap of the encoding sequence (GGGGSGGGCG, SEQIDNO:81) of the part of linker peptide and the 1-13 position residue of DDD2 is comprised by synthesis preparation two.Described oligonucleotide is annealed, and uses T4PNK phosphorylation, to form applicable and overhanging of being connected with the DNA that restriction endonuclease BamHI and PstI digests respectively 5 ' with 3 ' end.

on G4S-DDD2

5’GATCCGGAGGTGGCGGGTCTGGCGGAGGTTGCGGCCACATCCAGATCCCGCCGGGGCTCACGGAGCTGCTGCA-3’(SEQIDNO:82)

under G4S-DDD2

5’GCAGCTCCGTGAGCCCCGGCGGGATCTGGATGTGGCCGCAACCTCCGCCAGACCCGCCACCTCCG-3’(SEQIDNO:83)

Described double-stranded DNA and shuttle vectors CH1-DDD1-pGemT (continuous and meandering cross digest prepare) with BamHI and PstI are connected, to generate shuttle vectors CH1-DDD2-pGemT.Use SacII and EagI 507bp fragment to be cut from CH1-DDD2-pGemT, then this fragment is connected to IgG expression vector hMN14 (the I)-pdHL2 using SacII and EagI digestion preparation.Final expression construct is C-DDD2-Fd-hMN-14-pdHL2.

h679-Fd-AD2-pdHL2

H679-Fd-AD2-pdHL2 is the expression vector for generation of h679-Fab-AD2, and it has the anchoring domain sequence being connected to the AD2 of the C-terminal of CH1 by the Gly/Ser peptide linker of 14 amino-acid residues.AD2 respectively has a cysteine residues in the front and back of AD1 anchoring domain sequence.

As follows described expression vector is transformed.The complementary oligonucleotide (AD2 is upper with under AD2) of the encoding sequence of AD2 and the overlap of part linker sequence is comprised by synthesis preparation two.Described oligonucleotide is annealed, and uses T4PNK phosphorylation, form applicable and overhanging of being connected with the DNA that restriction endonuclease BamHI and SpeI digests respectively 5 ' with 3 ' end.

on AD2

5’GATCCGGAGGTGGCGGGTCTGGCGGATGTGGCCAGATCGAGTACCTGGCCAAGCAGATCGTGGACAACGCCATCCAGCAGGCCGGCTGCTGAA-3’(SEQIDNO:84)

under AD2

5’TTCAGCAGCCGGCCTGCTGGATGGCGTTGTCCACGATCTGCTTGGCCAGGTACTCGATCTGGCCACATCCGCCAGACCCGCCACCTCCG-3’(SEQIDNO:85)

Described duplex DNA is connected with shuttle vectors CH1-AD1-pGemT (preparing by digesting with BamHI and Spel), to generate shuttle vectors CH1-AD2-pGemT.Use SacII and EagI restriction enzyme to be cut from described shuttle vectors by 429 base pair fragment comprising CH1 and AD2 encoding sequence, then this fragment is connected to the h679-pdHL2 carrier using same enzyme digestion preparation.Final expression vector is h679-Fd-AD2-pdHL2.

The generation of embodiment 8.TF1

Carry out the extensive preparation of trivalent DNL construct (being called TF1) as follows.First according to stoichiometric concentration roughly, N-DDD2-Fab-hMN-14 (albumen L-purifying) and h679-Fab-AD2 (IMP-291-purifying) are mixed in 1mMEDTA, PBS, pH7.4.Before adding TCEP, SE-HPLC does not demonstrate a ₂any evidence (not shown) that b is formed.But have and represent a ₄(7.97 minutes; 200kDa), a ₂(8.91 minutes; 100kDa) and B (10.01 minutes; Peak 50kDa).Add 5mMTCEP and cause a fast ₂the formation of b mixture, consistent with 150kDa albumen, demonstrate this point (not shown) at the new peak of 8.43 minutes.Obviously there is excessive B in this experiment, because the peak corresponding to h679-Fab-AD2 (9.72 minutes) is still significantly, but correspond to a ₂or a ₄then have no obvious peak.Reduction reaction, after 1 hour, carries out dialysed overnight for the PBS changed several times, removing TCEP.Gained solution adds 10%DMSO and at room temperature preservation is spent the night.

When analyzing with SE-HPLC, represent a ₂the peak of b obviously comes to a point, and retention time slightly lower 0.1 minute, becomes 8.31 minutes (not shown)s, and according to the discovery that we are previous, this represents the raising of binding affinity.This mixture is further purified to remove κ chain impurity by IMP-291 affinity chromatography.As expected, excessive h679-AD2 is purified out together, then removes (not shown) with preparative SE-HPLC.

TF1 is high stability mixture.When measure TF1 and HSG (IMP-239) sensor chip in conjunction with time, response observed after Sample Injection terminates has no obvious reduction, by contrast, when measuring the solution containing C-DDD1-Fab-hMN-14 and h679-Fab-AD1 waiting molar mixture under simulated condition, during Sample Injection and after just having injected, the increase of viewed response unit, along with detectable decline, shows a starting to be formed ₂b structural instability.In addition, cause in for the response unit of TF1 significantly increase although inject WI2 subsequently, be not significantly increased for C-DDD1/AD1 mixture.

Because of WI2 and the extra increase of the response unit being fixed on the TF1 on sensor chip and being combined and cause, corresponding to two complete functional binding site, it is respectively caused by a subunit of N-DDD2-Fab-hMN-14 naturally.The binding ability of two Fab fragments of TF1 and WI2 confirms this point (not shown).After the mixture containing h679-AD2 and N-DDD1-hMN14 is correctly reduced and is oxidized into TF1, when analyzing with BIAcore, WI2, almost not additionally in conjunction with (not shown), shows a that disulfide linkage is stable ₂b mixture (such as TF1) is formed by means of only the interaction of DDD2 and AD2.

Two improvement are carried out to this process, to reduce time and the efficiency of process.First, with a ₄/ a ₂n-DDD2-Fab-hMN-14 and the h679-Fab-AD2 reaction of the molar excess a little that structure form of mixtures exists, makes not free h679-Fab-AD2, and is not bound to any a of h679-Fab-AD2 ₄/ a ₂structure and light chain, all use IMP-291 affinity chromatography to remove.The second, hydrophobic interaction chromatography (HIC) replaces dialysis or diafiltration, and for removing TCEP after reduction reaction, this not only shortens process time, but also adds the removal step of potential virus.N-DDD2-Fab-hMN-14 and 679-Fab-AD2 is mixed, at room temperature reduces 1 hour with 5mMTCEP.Add 0.75M ammonium sulfate to solution, be then loaded on ButylFFHIC post.Post is washed, to remove TCEP with the PBS containing 0.75M ammonium sulfate, 5mMEDTA.With PBS from wash-out reduction albumen HIC post, add 10%DMSO.At room temperature after overnight incubation, go out highly purified TF1 (Figure 22) with IMP-291 affinity protein purification.Without the need to extra purification step, example gel filters.

The generation of embodiment 9.TF2

After success produces TF1, C-DDD2-Fab-hMN-14 and h679-Fab-AD2 is reacted, also obtains a kind of analogue, be called TF2.Following generation has the TF2 of the pilot batch of the yield more than 90%.By the C-DDD2-Fab-hMN-14 (200mg) of albumen L-purifying and h679-Fab-AD2 (60mg) with the mixed in molar ratio of 1.4:1.Containing in the PBS of 1mMEDTA, total protein concentration is 1.5mg/ml.Subsequent step comprises TCEP reduction, HIC chromatography, DMSO oxidation and IMP-291 affinity chromatography, identical all with for described in TF1.Before adding TCEP, SE-HPLC does not demonstrate a ₂any evidence (not shown) that b is formed.But exist corresponding to a ₄(8.40 minutes; 215kDa), a ₂(9.32 minutes; 107kDa) and b (10.33 minutes; Peak 50kDa).Add 5mMTCEP and cause a fast ₂the formation of b mixture, (not shown) as the new peak at 8.77 minutes proves, this peak is consistent with the 157kDa albumen desired by diadactic structure.By IMP-291 affinity chromatography, TF2 is purified to close to homogeneous (not shown).From product, a is eliminated to the SE-HPLC analytical proof that IMP-291 carries out in conjunction with fraction ₄, a ₂with free κ chain (not shown).

The function of TF2 is measured according to for the BIACORE that utilizes described in TF1.By TF2, C-DDD1-hMN-14+h679-AD1 (as non-covalent a ₂the control sample of b mixture) or C-DDD2-hMN-14+h679-AD2 (as non-reduced a ₂with the control sample of b component) be diluted to 1 μ g/ml (gross protein) and sensor chip by securing HSG.Be about 2 times of two control sample reactions to the response of TF2, show in control sample, only have h679-Fab-AD component be combined with sensor chip and retain thereon.Inject WI2IgG subsequently, the DDD-Fab-hMN-14 component proving only have TF2 to have and combine closely with h679-Fab-AD, shown in extra signal response.Because WI2 increases with the response unit being fixed on the TF2 on sensor chip and being combined and cause, also correspond to two complete functional binding site, each is caused by C-DDD2-Fab-hMN-14 subunit.The binding ability of two Fab fragments of TF2 and WI2 confirms this point (not shown).

The relative CEA-binding affinity of TF2 is measured by competitive ELISA.With the fusion rotein bag of the A3B3 structural domain containing CEA by each plate (0.5 μ g/ hole), described albumen is identified by hMN-14.Serial dilution TF1, TF2 and hMN-14IgG, in quadruplicate, containing put together HRP hMN-14IgG (1nM) hole in incubation.Data presentation TF2 with at least equal avidity of the avidity with IgG in conjunction with CEA, 2 times (not shown)s stronger than TF1.

The serum stability of embodiment 10.TF1 and TF2

TFl and TF2 is designed to the stable constraint structure that can be used in body, wherein Macrodilution can occurs in blood and tissue.By the stability of TF2 in BIACORE evaluator serum.TF2 Freshman serum (merge from 4 donors and obtain) is diluted to 0.1mg/ml and at 5%CO ₂, 37 DEG C of incubations 7 days.Every day, sample diluted by 1:25, was then analyzed by BIACORE, used IMP-239HSG sensor chip.Injection WI2IgG, and the amount of TF2 of complete activity complete for quantitative assay.By serum sample compared with the control sample directly diluted from stoste.TF2 stablizes at serum camber, within 7 days, keeps its dual specific binding activities (not shown) of 98% afterwards.In people or mice serum, similar result (not shown) is observed for TF1.

The generation of embodiment 11.C-H-AD2-IgG-pdHL2 expression vector

PdHL2 mammalian expression vector has been used to mediate expression people such as (, Methods2005,36:84-95) Qu of many restructuring IgG.It is C-H-AD2-IgG-pdHL2 carrier that generation plasmid shuttle vector is beneficial to any IgG-pdHL2 vector.Use pdHL2 carrier is as template and oligonucleotide FcBglII is left and the right gene as primer amplification Fc (CH2 and CH3 structural domain) of FcBam-EcoRI.

fcBglII is left

5’-AGATCTGGCGCACCTGAACTCCTG-3’(SEQIDNO:86)

fcBam-EcoRI is right

5’-GAATTCGGATCCTTTACCCGGAGACAGGGAGAG-3’(SEQIDNO:87)

Amplimer is cloned into pGemTPCR cloning vector.Use XbaI and BamHI restriction enzyme to cut Fc inset fragment from pGemT, be then connected to the AD2-pdHL2 carrier prepared with XbaI and BamHI digestion h679-Fab-AD2-pdHL2, to produce shuttle vectors Fc-AD2-pdHL2.

In order to any IgG-pdHL2 expression vector is changed into C-H-AD2-IgG-pdHL2 expression vector, cut 861bpBsrGI/NdeI restriction fragment from the former, replaced with the 952bpBsrGI/NdeI restriction fragment cut from Fc-AD2-pdHL2 carrier.BsrGI in CH3 structural domain internal cutting and NdeI cut in the downstream (3 ') of expression cassette.

The generation of embodiment 12.C-H-AD2-hLL2IgG

Epratuzumab (or hLL2IgG) is humanization Anti-Human CD22MAb.As described in example 11 above, from the expression vector that hLL2IgG-pdHL2 produces C-H-AD2-hLL2IgG, transfection Sp2/0 myeloma cell is used it for by electroporation.After transfection, be coated with 96 orifice plates with described cell, in containing the substratum of methotrexate, select transgene clone.The C-H-AD2-hLL2IgG productive rate of the detection screening and cloning of the sandwich ELISA being coated with the microtiter plate of the anti-idiotype MAb of hLL2-specificity by use and the Anti-Human IgG puting together peroxidase.By clonal expansion to rolling bottle to prepare protein, and from the substratum purifying C-H-AD2-hLL2IgG exhausted in the single stage using protein-A affinity chromatography.SE-HPLC analyzes and carries out parsing (not shown) to two protein peaks.The retention time (8.63 minutes) of slow elution peak is similar to hLL2IgG.The retention time (7.75 minutes) of fast elution peak corresponds to about 300kDa protein.Determined that this peak represented the disulfide linkage connection dimer of C-H-AD2-hLL2-IgG afterwards.This dimer is reduced to monomeric form in DNL reaction.SDS-PAGE analyzes and shows, the dimeric forms that the C-H-AD2-hLL2-IgG of purifying is connected with disulfide linkage by two kinds of monomers of module forms (not shown).The protein band representing these two kinds of forms is high-visible in the SDS-PAGE under non-reducing conditions, but under the reducing conditions, form of ownership is all reduced to two the band (not shown)s representing composition polypeptide (heavy chain-AD2 and κ chain).Do not detect that other pollutes band.

The generation of embodiment 13.C-H-AD2-hA20IgG

HA20IgG is humanization anti-humen CD 20 MAb.Described in embodiment 27, produce the expression vector of C-H-AD2-hA20IgG from hA20IgG-pDHL2, then this carrier is passed through electroporation transfection Sp2/0 myeloma cell.After transfection, be coated with 96 orifice plates with described cell, and screen transgene clone on the substratum comprising methotrexate.The C-H-AD2-hA20IgG productive rate of the detection screening and cloning of the anti-human igg of peroxidase is puted together by the sandwich ELISA and use using the 96 hole microtiter plates of coating hA20 specific anti-idiotypic MAb.By clonal expansion to rolling bottle to prepare protein, and it is closely similar to analyze the result of the C-H-AD2-hLL2IgG obtained in the result and embodiment 28 obtained from the substratum purifying C-H-AD2-hA20IgG exhausted, SE-HPLC and SDS-PAGE in the single stage using protein-A affinity chromatography.

The generation of Fab and the IgG fusion rotein from multiple antibody that embodiment 14.AD-is connected with DDD-

By using the technology described in previous embodiment, building following IgG or Fab fusion rotein, and being integrated into DNL construct.Described fusion rotein keeps the antigen binding characteristics of parental generation antibody and DNL construct shows integrated antibody or the antigen-binding activity of antibody fragment.

Table 2. comprises the fusion rotein of IgG or Fab part

Fusion rotein	Binding specificity
		C-AD1-Fab-h679	HSG
C-AD2-Fab-h679	HSG
		C-(AD2) 2-Fab-H679	HSG
C-AD2-IgG-h734	Indium-DTPA
		C-AD2-IgG-hA20	CD20
C-AD2-IgG-hA20L	CD20
		C-AD2-IgG-hL243	HLA-DR
C-AD2-IgG-hLL2	CD22
		N-AD2-IgG-hLL2	CD22
C-AD2-IgG-hMN-14	CEA
		C-AD2-IgG-hR1	IGF-1R
C-AD2-IgG-hRS7	EGP-1
		C-AD2-IgG-hPAM4	MUC1
C-AD2-IgG-hLL1	CD74
C-DDD1-Fab-hMN-14	CEACAM5
		C-DDD2-Fab-hMN-14	CEACAM5
C-DDD2-Fab-h679
		C-DDD2-Fab-hA19	CD19
C-DDD2-Fab-hA20	CD20
		C-DDD2-Fab-hAFP	AFP

C-DDD2-Fab-hL243	HLA-DR
		C-DDD2-Fab-hLL1	CD74
C-DDD2-Fab-hLL2	CD22
		C-DDD2-Fab-hMN-3	CEACAM6
C-DDD2-Fab-hMN-15	CEACAM6
		C-DDD2-Fab-hPAM4	MUC1
C-DDD2-Fab-hR1	IGF-1R
		C-DDD2-Fab-hRS7	IGP-1
N-DDD2-Fab-hMN-14	CEACAM5

Embodiment 15. is based on the generation of the DDD module of Interferon, rabbit (IFN)-α 2b

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

Utilize the cDNA sequence of PC amplification IFN-α 2b, thus generation is included in the sequence of its C-terminal fusion to the IFN-α 2b of the polypeptide of one sequence:

KSHHHHHHGSGGGGSGGGCGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQIDNO:88).

Use total length people IFN α 2bcDNA clone (InvitrogenUltimateORF people clones cat#HORF01CloneIDIOH35221) as template and following oligonucleotide as primer to realize pcr amplification:

iFNA2Xba is left

TCTAGACACAGGACCTCATCATGGCCTTGACCTTTGCTTTACTGG(SEQIDNO:89)

iFNA2BamHI is right

GGATCCATGATGGTGATGATGGTGTGACTTTTCCTTACTTCTTAAACTTTCTTGC(SEQIDNO:90)

Pcr amplification primer is cloned into pGemT carrier.Following preparation DDD2-pdHL2 mammalian expression vector is to be connected with IFN-α 2b.XbaI and BamHI is used to digest C _h1-DDD2-Fab-hMN-14-pdHL2 (people such as Rossi, ProcNatlAcadSciUSA2006,103:6841-6) carrier, this removes all Fab gene orders but leaves DDD2 encoding sequence.Use XbaI and BamHI to cut IFN-α 2b amplimer from pGemT, then connect into DDD2-pdHL2 carrier to produce expression vector IFN-α 2b-DDD2-pdHL2.

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

IFN-α 2b-DDD2-pdHL2 SalI digests and linearized, then by electroporation method transfection Sp/ESF myeloma cell.Finding that two clones have by ELISA can the IFN-α 2b of detection level.(called after 95) adapts to growth in serum free medium and reduces without significant productive rate one of to make two to clone.Cloned with the MTX concentration the increased progressively amplification from 0.1 to 0.8 μM in 5 weeks subsequently.In this stage, carry out subclone by limiting dilution to it, amplification has the subclone (95-5) of production peak.Use business rIFN-α 2b (ChemiconIF007, Lot06008039084) as standard, the productive rate of the 95-5 grown in shaking flask is 2.5mg/L according to estimates.

From the batch culture purifying IFN-α 2b-DDD2 cultivated rolling bottle

Clone 95-5 is equipped with in the rolling bottle of Serum-free Hybridoma SFM and the 0.8 μM MTX of 20L altogether at 34 and increases, reached end eventually and cultivate.Following process nutrient solution, then utilizes immobilized metal affinity chromatography (IMAC) purifying IFN-α 2b-DDD2.By centrifugal clarification supernatant liquor, carry out 0.2 μM of filtration, diafiltration is to 1X binding buffer liquid (10mM imidazoles, 0.5MNaCl, 50mMNaH ₂pO ₄, pH7.5) in, be concentrated into 310mL, add the final concentration of Tween20 to 0.1%, then add on 30-mLNi-NTA post.After introduction of the sample, wash pillar with the Tween20 in 1X binding buffer liquid of 500mL0.02%, use the 30mM imidazoles of 290mL, 0.02%Tween20,0.5MNaCl, 50mMNaH subsequently ₂pO ₄, pH7.5 washs.With 250mM imidazoles, 0.02%Tween20,150mMNaCl, 50mMNaH of 110mL ₂pO ₄, pH7.5 eluted product.Be purified into the IFN α 2b-DDD2 of about 6mg.

IFN-α 2b-DDD2 is produced in Bacillus coli cells

IFN-α 2b-DDD2 is expressed as soluble protein by fermentable in intestinal bacteria.Use IFN-α 2b-DDD2-pdHL2DNA as template, by pcr amplification encoding gene.Use NdeI and XhoI restriction site that amplimer is cloned into pET26b coli expression carrier.By inducing LB shaking flask to carry out carrying out cell inner expression protein in BL21pLysS host cell in 12 hours with 100 μMs of IPTG at 18 DEG C.Utilize IMAC from cell lysate purification of soluble IFN-α 2b-DDD2 as mentioned above.

Embodiment 16. comprises and is connected to C _h3the generation of the DNL conjugate of the IFN-α 2b-DDD2 of-AD2-IgG

(what comprise 4 copies is connected to a C to the DNL construct of called after 20-2b _h3the IFN α 2b-DDD2 of-AD2-IgG) pass through two DNL module C by DNL _h3the combination of-AD2-IgG-v-mab and IFN α 2b-DDD2 produces, and expresses in each comfortable Sp/ESF of described DNL module.By the similar names had for 20-2b (humanization IgG1+4IFN α 2b) but have different target Mab other DNL-produce MAb-IFN α construct in several experiment with comparing: 22-2b is with C _h3-AD2-IgG-e-mab (epratuzumab) as its AD2 module, the anti-CD22 of this module and in conjunction with lymphoma; 734-2b is with C _h3-AD2-IgG-h734 as its AD2 module, this module antiheparin, In-DTPA and not in conjunction with any animal protein or tissue; And R1-2b uses C _h3-AD2-IgG-hR1, it is in conjunction with human insulin-like growth factor 1 acceptor (IGF-1R).

Following preparation 20-2bDNL construct.By the C selected _h3-AD2-IgG combines with the IFN-α 2b-DDD2 of about 2 molar equivalents, after adding 1mMEDTA and 2mM reduced glutathion (GSH), at room temperature go back original mixture in a mild condition.Add Sleep-promoting factor B to 2mM, make mixture at room temperature keep other 12-24 hour.DNL conjugate is carried out purifying on albumin A affinity column.Preparation be called 20-2b, 22-2b, hR1-2b and 243-2b 4 kinds of these type of DNL conjugates (each self-contained 4 copy be anchored on C respectively _h3-AD2-IgG-hA20 (there is the specificity to CD20), C _h3-AD2-IgG-hLL2 (there is the specificity to CD22), C _h3-AD2-IgG-hR1 (there is the specificity to IGF-1R) and C _h3iFN-α 2b on-AD2-IgG-hL243 (there is the specificity to HLA-DR)).Display is separately analyzed to the SE-HPLC of the 20-2b of the IFN-α 2b-DDD2 produced from Mammals (m) or intestinal bacteria (e) there is the main peak (not shown) of the retention time consistent with the covalent complex be made up of IgG and 4 IFN-α 2b group.Similar SE-HPLC characteristic spectrum is observed for other 3 IFN-IgG conjugates.

The external activity of IFN-IgG conjugate

Use and with lymphoma proliferation assay, the Bioactivity of the external IFN α biological activity of 20-2b with business PEGization IFN α 2 reagent PEGASYS with PEG-Intron is compared based on report of cell, viral protection.Use the kit measurement specific activity based on cell, described test kit uses the transgenic human premonocyte system (Figure 1A-1D) of the reporter gene carrying the response element merged to Interferon, rabbit stimulation.The specific activity (5300IU/pmol) of 20-2b is greater than PEGASYS (170IU/pmol) and PEG-Intron (3400IU/pmol) (Figure 1A).Be similar to 734-2b, 1R-2b and 5 other MAb-IFN α constructs (data do not show) specific activity (4000-8000IU/pmol) that display is similar separately of 20-2b generation, this proves the consistence (Figure 1A) for generation of the DNL method of this class formation.4 IFN α 2b groups all cause the effect of the MAb-IFN α of enhancing.When for IFN α equivalent stdn, specific activity/IFN α is about 10 times of PEGASYS and is only 1/2 of PEG-Intron.

MAb-IFN α, PEGASYS and PEG-Intron in vitro virus protection measure in comparison prove that MAb-IFN α keeps IFN α 2b antiviral activity, have similar to PEG-Intron and be the specific activity (Figure 1B) of about 10 times of PEGASYS.

IFN α 2b can have direct antiproliferative or cytotoxic effect to some tumour strain.In vitro in proliferation assay, utilize the activity (Fig. 1 C) the extremely sensitive Burkitt lymphoma cell line of IFN α (Daudi) being measured to 20-2b.Each IFN α 2 reagent is in vitro with efficient (EC ₅₀=4-10pM) suppress (>90%) Daudi efficiently.But, 20-2b (EC ₅₀=0.25pM) effect be about 30 times of non-targeted MAb-IFN α construct.

The parental generation anti-CD 20 MAb of 20-2b is in vitro at significantly higher concentration (EC ₅₀>10nM) to many lymphoma cell lines (comprising Daudi (people such as Rossi, 2008, CancerRes68:8384-92)), there is anti-proliferation activity on.

Also use Jeko-1 to assess the external activity of 20-2b, described cell is the lymphoma mantle cell strain (Fig. 1 D) IFN α and anti-CD 20 to lower susceptibility.Jeko-1 only has appropriate susceptibility to parental generation anti-CD 20 MAb, has the maximum suppression (I of 10% _max), EC ₅₀be about 1nM.As shown in for 734-2b, Jeko-1 (I _max=43%; EC ₅₀=23pM) Daudi (I is not so good as to the reaction of IFN α 2b _max=90%; EC ₅₀=7.5pM) strong.Compared with 734-2b, 20-2b is (I in larger degree _max=65%) suppress Jeko-1 and show dimorphism dose response curve (Fig. 1 D).When concentration <10pM, the lower concentration observed owing to IFN α 2b reacts, its I _max=43% reaches platform, similar to 734-2b.In the reaction of more than 100pM high density clearly, now I _maxreach 65%.The lower concentration IFN α 2b of 20-2b reacts (EC ₅₀=0.97pM) effect be 25 times of 734-2b, similar to the result for Daudi.

Accumulation/synergistic effect that the combination measuring parental generation anti-CD 20 antibodies and 734-2b (v-mab+734-2b) is conducted owing to CD20 and IFN signal α with the effect illustrating the enhancing of 20-2b.Except the concentration of >1nM (wherein for the former suppress increase but for the latter's not this effect), the dose response curve of v-mab+734-2b is similar substantially to independent 734-2b.These results show that MAb target is the lower EC causing 20-2b ₅₀reason, but its larger I _maxobviously owing to the cumulated activity of IFN α 2b and CD-20 intracellular signaling.The effect of CD20 intracellular signaling only reacts (EC in the high density of 20-2b ₅₀=0.85nM) in be significant, this effect and reaction (EC to v-mab ₅₀=1.5nM) quite.Unconspicuous for v-mab+734-2b dimorphism dose response curve, because two reactions are overlapping.But storage effect is significant when concentration >1nM.The I of 20-2b _max(65%) than IFN α 2b (I _max=43%) and v-mab (I _max=10%) accumulation reaction is large, may there is synergy between the effect which imply that IFN α 2b and v-mab.

ADCC is active

IFN α strengthens ADCC activity by activation NK cell and scavenger cell, and this is the basic role mechanism (MOA) of anti-CD 20 immunotherapy.We use peripheral blood lymphocytes (PBMC) action effect cell, utilize two NHL cells to compare the ADCC of 20-2b and v-mab.Use from the PBMC of multiple donor, replication as one man shows 20-2b and v-mab and compares and have the ADCC of enhancing, as (Fig. 3 A) as shown in for Daudi and Raji cell.This effect be also show for 22-2b (comprising the MAb-IFN α of anti-CD22MAb), epratuzumab, ADCC people such as (, 2007, MolImmunol44:1331-41) Carnahan of its display appropriateness.

CDC is active

CDC is considered to the important MOA of I type anti-CD 20 MAb (comprising v-mab and Rituximab).But, lack this function people such as (, 2002, CancerImmunolImmunother51:15-24) Cardarelli in II type MAb (being represented by tositumomab), but it has anti-anti-lymphoma effects.Different from v-mab, 20-2b does not show CDC activity (Fig. 3 B) in vitro.These results with based on C _h3the result of other DNL of-AD2-IgG-v-mab module is consistent, and wherein combine may because of spatial interference obviously impaired people such as (, 2008) Rossi for complement.

The pharmacokinetics (PK) of embodiment 17.20-2b is analyzed

Pharmacokinetics (PK) character of 20-2b is assessed in Male Swiss-Webster mice, by itself and PEGASYS, PEG-INTRON and α 2b-413 (PEGization IFN prepared by DNL, see the 11/925th, No. 408 U.S. Patent application) pharmacokinetic property compare.Utilize the IFN-α concentration in serum sample in ELISA mensuration different time points.The specific activity of iLiteHumanInterferonAlphaCell-BasedAssay kit measurement IFN α 2b is used according to the scheme (PBLInterferonSource) of manufacturers's suggestion.Fig. 2 represents the result that PK analyzes, and its display 20-2b has the elimination significantly slower than other reagent and longer serum stores.When the injected dose of 210pmol, be 8.0 hours (20-2b), 5.7 hours (α 2b-413), 4.7 hours (PEGASYS) and 2.6 hours (PEG-INTRON) with the pharmacokinetics serum half-life as calculated hour to represent.Elimination factor (1/h) is 0.087 (20-2b), 0.121 (α 2b-413), 0.149 (PEGASYS) and 0.265 (PEG-INTRON).MRT as calculated _0.08→ ∞ (hr) is 22.2 (20-2b), 12.5 (α 2b-413), 10.7 (PEGASYS) and 6.0 (PEG-INTRON).Because by mixture but not the property testing pharmacokinetic parameter of individual antibody or cytokine, therefore expect that the PK feature of cytokine-DNL mixture is attributable to other cytokine moiety and antibody moiety and is not limited to above-mentioned specific 20-2b construct.

The activity in vivo of embodiment 18.20-2b

Serum stability

20-2b is stable (not shown) at 37 DEG C in human serum (>=10 days) or whole blood (>=6 days).Dual specific ELISA assay method is used to measure the concentration of 20-2b mixture.Along with in past in the period whole blood measured or serum, serum 20-2b level there is no detectable change.

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

We compare 20-2b, v-mab, 734-2b or v-mab+734-2b eliminate lymphoma or normal B cells in ex vivo environment ability (Fig. 4) from whole blood.The therapeutic efficiency of naked anti-CD 20 MAb it is believed that and realizes people such as (, 2007, MolImmunol44:3823-37) Glennie by the apoptosis of 3 mechanism of action (MOA)-intracellular signaling induction or cessation of growth cessation, ADCC and CDC.In this mensuration, v-mab can utilize all 3 MOA, and simultaneously based on external discovery, 20-2b potentially can utilize intracellular signaling and strengthen ADCC but non-CDC.In this short-run model, the IFN α 2b group of 20-2b and 734-2b can directly act on tumour cell, and the ADCC strengthening v-mab is active, and may have certain immunostimulation.But, in this isolated measuring of 2 days, fail the full spectrum of activation of the IFN α-mediation obtaining the natural immune system that may occur in vivo and adaptive immune system.

On 0.01nM, 20-2b gets rid of energy force rate v-mab (22.8%), 734-2b (38.6%) or the v-mab+734-2b (41.7%) significantly stronger (Fig. 4) of Daudi cell (60.5%).On 0.1nM, 20-2b with v-mab+734-2b consumes Daudi with similar degree (88.9%), and this is than v-mab (82.4%) or 734-2b (40.7%) stronger (Fig. 4).On 1nM, except 734-2b (55.7%), the Daudi (Fig. 4) of consumption 95% got rid of by each reagent.Each difference though statistically significant (P<0.01) of instruction.

Ramos and Daudi compares not too responsive to IFN α 2b and v-mab.The effect of 734-2b is only medium, each concentration causes the Ramos being less than 20% consume (Fig. 4).On 0.01 and 0.1nM, 20-2b and v-mab+734-2b compare consume more Ramos, v-mab+734-2b consume more cell (Fig. 4) than v-mab successively.On 1nM, except 734-2b, all process all cause similar Ramos to consume (75%) (Fig. 4).Each difference though statistically significant (P<0.02) of instruction.

As shown in for 734-2b, independent IFN α 2b does not get rid of normal B cells in this mensuration.On these lower concentrations, 20-2b, v-mab and v-mab+734-2b show the dose response consumption of similar B cell separately, and it is significantly lower than the consumption to Daudi or Ramos.Neither one process causes the remarkable consumption (data do not show) of T cell.

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

The restriction of mouse model is the pole Wheat Protein of mouse cell to people IFN α 2b.The enhancing of the natural immunity and adaptive immunity can be comprised in the overall therapeutic of the 20-2b that can realize in people is favourable.Remember that these limit, effect in the anti-lymphoma body that we study 20-2b anti-dissemination Burkitt lymphoma model in SCID mouse.In our initial Daudi model in early days, test is to high susceptibility (Fig. 5 A).Second day after inoculation, use 20-2b, v-mab or 734-2b of single low dosage to group.When (contrasting for irrelevant MAb-IFN α for v-mab with salt solution, when 734-2b) comparing, what v-mab or 734-2b of the single dose of 0.7pmol (170ng) caused survival rate significantly improves (P<0.0001) (Fig. 5 A).This raising is appropriate, and median overall survival (MST) is increased to 34 days (for v-mab) from 27 days (for salt solution).But MST is increased more than 100 day (P<0.0001) (Fig. 5 A) relative to saline control and v-mab group by the 20-2b of the single dose of 0.7pmol (170ng).After 19 weeks, terminate research, 7 long-term survivorses (LTS) now in 0.7pmol20-2b treatment group do not find the tangible proof (healing) (Fig. 5 A) of disease through postmortem.Surprisingly, even the 20-2b of the lowest dose level of 0.07pmol (17ng) also makes MST extend more than 2 times (Fig. 5 A).

Next, we assess effect of 20-2b in Daudi model having more challenging late period, in described model, make mouse produce significantly larger tumor load (Fig. 5 B) before treatment.After tumor inoculation 7 days, use 20-2b, v-mab, 734-2b or PEGASYS of single low dosage (0.7,7.0 or 70pmol) to group.The MST of saline control mouse is 21 days (Fig. 5 B).PEGASYS or 734-2b of maximum dose level (70pmol) (it has the Pk (compared with restructuring IFN α 2b) of enhancing but not target tumor separately) makes MST double (42 days; P<0.0001) (Fig. 5 B).Result (38.5 days) (Fig. 5 B) that utilize the process generation of the 20-2b of the dosage of 1/100 (0.7pmol) similar to PEGASYS or 734-2b of maximum dose level (70pmol).Utilize the process of the 20-2b of the dosage of 1/10 (7pmol) to cause and utilize survival time (80.5 days, 20%LTS) (P<0.0012) (Fig. 5 B) that significantly improve compared with the process of PEGASYS or 734-2b of 70pmol.On the maximum dose level (70pmol) of test, 20-2b makes the MST of 100%LTS be increased to more than 105 days (Fig. 5 B).We had previously utilized early stage model proof v-mab above can increase the survival time of lotus Daudi mouse at relatively low dosage (3.5pmol), and higher dosage causes LTS.But in this late stage tumor model, the v-mab of the 70pmol of single dose only has medium effect (although being significant) (MST=24 days, P=0.0001) (Fig. 5 B) to the survival time.

We measure 20-2b having more in challenging model subsequently, and the direct suppression that described model causes IFN α compared with Daudi is not too responsive and to utilizing the immunotherapy of v-mab to react not too strong.Compared with Daudi, the direct acting susceptibility of Raji to IFN α 2b is about 1/100.But Raji and Daudi has similar CD20 antigen density (people such as Stein, 2006, Blood108:2736-44) and react to v-mab, although obviously (people such as Goldenberg, 2009 strong not as Daudi, Blood113,1062-70).Effect of 20-2b is studied late, 5 days begin treatments (Fig. 6 A) after tumor inoculation in Raji model.6 injections (each 250pmol) are altogether used to group within the time of 2 weeks.734-2b does not increase the survival time (MST=16 days) relative to salt solution, consistent with the insensitivity of Raji to IFN α (Fig. 6 A).V-mab significantly increases the survival time (MST=26 days, P<0.0001) (Fig. 6 A) relative to salt solution.20-2b is better than every other process (MST=33 days, P<0.0001) (Fig. 6 A).

Finally, we utilize NAMALWA, there is the low direct acting susceptibility to IFN α (there is the CD20 antigen density of about 1/25 compared with Daudi or Raji and be considered to there is the resistance (people such as Stein to anti-CD 20 immunotherapy, 2006) human lymphoma), effect (Fig. 6 B) of research 20-2b.20-2b or 734-2b of 6 dosage (each dosage 250pmol) is altogether used to group.The v-mab of 7 dosage (each dosage 3.5nmol) is altogether used to another group.The group of brine treatment is utilized to have the MST (Fig. 6 B) of 17 days.The process of 734-2b is utilized (although significantly) to increase the survival time (MST=20 days, P=0.0012) (Fig. 6 B) as mild as a dove.20-2b (MST=34 days) is better than the 734-2b (P=0.0004) that gives with 14 multiple doses and v-mab (MST=24 days, P=0.0026) (Fig. 6 B).

Conclusion

The target that result shows the IFN α with anti-CD 20 MAb clearly makes immune cell factor have more effect and efficiency than alone or in combination reagent.The MAb target of IFN α to tumour can allow the dosage regimen of carrying out the lower single agents of frequency, reduces or eliminates the side effect relevant to IFN therapy, and causes effect of significantly enhancing.In addition, target IFN α can the mediation of inducing acute tumour immune response and can stimulate by the pleiotropy of the natural immunity and adaptive immunity and cause the immunological memory (people such as Belardelli, 2002, CytokineGrowthFactorRev12:119-34).Other research groups have produced the MAb-IFN α by chemically conjugated preparation, and described MAb-IFN α shows some potential clinical benefit (people such as Pelham, 1983, CancerImmunolImmunother15:210-16 of this type of construct; The people such as Ozzello, 1998, BreastCancerResTreat48:135-47).The restructuring MAb-IFN α comprising mouse IFN α and anti-HER2/neuMAb shows the strong suppression of transgenosis (HER2/neu) mouse B cell knurl and can by the immunological memory eliciting protective adaptive immunity reaction (people such as Huang in immunocompetent mouse; 2007, JImmunol179:6881-88).

We utilize the treatment of 20-2b will stimulate local recruitment and the activation of many immune cell (comprising NK, T4, T8 and dendritic cell) at expection, thus cause strengthen cytotoxicity and ADCC, and can potentially induced tumor mediation immunological memory.But, mouse cell compared with people's cell to people IFN α 2b extremely insensitive (logarithmic value is about 4) (people such as Kramer, 1983, JInterferonRes3:425-35; The people such as Weck, 1981, JGenVirol57:233-37).Therefore, the IFN α 2b almost not having the anti-anti-lymphoma effects of (if any) 20-2b to be attributable to mouse immune reaction in above-mentioned mouse model In vivo study activates.On the contrary, kill and wound main owing to IFN α 2b to the direct effect of lymphoma cell.

We have shown the ADCC that 20-2b has enhancing, and this can be the most important MOA of anti-CD 20 immunotherapy.But because people IFN α 2b is the stimulant of very weak mouse host effector cell, therefore when it is present in people, the ADCC of IFN α-enhancing may be not accomplished.Even if having these restrictions, in body, result still shows 20-2b can be Effective Anti-lymphoma reagent, is shown as the effect of 100 times of v-mab or non-targeted MAb-IFN α in IFN α-susceptibility Daudi model.Even if for the direct effect of IFN α relatively not too responsive (Raji/NAMALWA) or lymphoma model anti-CD 20 immunotherapy (NAMALWA) being had to resistance, 20-2b still shows the effect being better than v-mab or non-targeted MAb-IFN α.

The fusion of IFN α 2b to v-mab strengthens effect in its body by extending cycling time and making it possible to cancer target.The therapeutic potential of Pk is proven in Daudi model, more slowly removes EGASYS and be better than removing PEG-Intron quickly in described model, and it has higher activity specific (data do not show).20-2b, obviously than PEGASYS or 734-2b more effective force, to show by the lymphoma target of anti-CD 20 MAb for the effect of its excellence and effect it is vital.Surprisingly, even if the impact of target is also obvious in measuring in vitro.In vitro in proliferation experiment (only allow carry out lymphoma suppression by intracellular signaling), 20-2b and non-targeted MAb-IFN α show activity compared with (individually or when combining with v-mab) in the concentration of 1/25.Ex vivo environment allows all 3 anti-CD 20 MOA all to work.Even if active without CDC, 20-2b is also more efficient than IFN α or v-mab (either individually or in combination) in the lymphoma getting rid of to come autoblood, this demonstrate that the importance of target.In in vitro/vitro study, the impact of MAb target is somewhat astonishing, because MAb, effector and target cell are all limited in whole experimentation.We expect that 20-2b affects having in significantly larger body in people patient.

The IFN α 2b of 20-2b and v-mab component significantly with accumulation or cooperative mode effect, can cause the effect that it strengthens.In-vitro multiplication measures and shows to exist at least storage effect, and this is confirmed by the result that the combination of wherein v-mab and 734-2b is better than the vitro study of arbitrary independent reagent.This ADCC by the increase of the v-mab of the part as 20-2b or realize in vitro when combining with 734-2b, but ADCC does not have function in proliferation assay in vitro, this shows to there is other mechanism.The signal of the CD20 conduction combined by v-mab-can increase IFN signal α, thus causes the effect of enhancing.Selectively, as in slowly and the combination of the v-mab of MAb can stop the internalization/downward of I type IFN acceptor, thus cause the signal of longer time and more effective IFN α-induction.

Embodiment 19. is based on the generation of the DDD module of erythropoietin (EPO)

For the structure of EPO-DDD2-pdHL2 of expressing in mammalian cell

By the cDNA sequence of pcr amplification EPO, produce the EPO merging the polypeptide be extremely made up of following sequence at its C-terminal:

KSHHHHHHGSGGGGSGGGCGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQIDNO:88)

Total length people PCREPOcDNA clone is used to carry out pcr amplification as template and following oligonucleotide as primer:

ePOXbaI is left

TCTAGACACAGGACCTCATCATGGGGGTGCACGAATGTCC(SEQIDNO:91)

ePOBamHI is right

GGATCCATGATGGTGATGATGGTGTGACTTTCTGTCCCCTGTCCTGCAG(SEQIDNO:92)

Pcr amplification primer is cloned into pGemT carrier.Preparation DDD2-pdHL2 mammalian expression vector, by being connected it with EPO with the digestion of XbaI with BamHI restriction endonuclease.Use XbaI and BamHI to cut EPO amplimer from pGemT, connect into DDD2-pdHL2 carrier to produce expression vector EPO-DDD2-pdHL2.

The mammalian cell expression of EPO-DDD2

EPO-pdHL2 SalI digest and linearized, then by electroporation method, it is stably transfected into Sp/ESF myeloma cell.Clone is selected with the substratum containing 0.15 μM of MTX.Catch the ELISA of the fusion rotein of His-mark by using NuncImmobilizer nickel-inner complex plate and utilize the detection display of anti-EPO antibody clone #41,49 and 37 to produce the EPO of about 0.5mg/L separately.IMAC is utilized to be purified into the EPO-DDD2 of about 2.5mg from 9.6 liters of serum-free spinner culture things.

Embodiment 20.734-EPO, comprises 4 and is connected to C _h3the generation of the DNL conjugate of the EPO-DDD2 part of-AD2-IgG-h734

As above for the generation 734-EPO described by 20-2b.The SE-HPLC of the 734-EPO of albumin A-purifying analyzes main peak and the shoulder (not shown) of the larger molecular size of display.The retention time of main peak is consistent with by IgG and 4 covalent complex that EPO group forms.Shoulder may owing to the non-covalent dimer of IgG-EPO conjugate.Under utilizing the DS-PAGE of Coomassie blue stain analysis and anti-EPO immunoblotting assay to be presented at non reducing conditions, described product has the Mr (not shown) being greater than 260kDa, consistent with the MW of the about 310kDa derived.Under the reducing conditions, the band representing 3 composition polypeptide (EPO-DDD2, heavy chain-AD2 and light chain) of 734-EPO is high-visible and in amount, seem similar (not shown).Non-product pollution thing do not detected.

Recombinant human epo (Calbiochem) is used to measure the ability (ATCC) of the reactive TF1 Growth of Cells of stimulation EPO-of EPO-DDD2 and 734-EPO as positive control.TF1 cell is being contained 1 × 10 ⁴cultivate in 96 orifice plates of individual cells/well in the RPMI1640 substratum supplemented without GM-CSF being supplemented with 20%FBS.Commercial reagents box (HumanerythropoietinELISA test kit, StemCellResearch, Cat#01630) is used to measure the concentration (unit/ml) of EPO construct.Deposit in case with the concentration culturing cell of 900u/ml to 0.001U/ml at rhEPO, EPO-DDD2 or 734-EPO, carry out 72 hours.Before being to measure OD490 in 96 orifice plates reading meters, using 20 μ lMTS reagent/hole incubation 6 hours to be measured by MTS and compare viable cell density.GraphPadPrism software is used to produce dose response curve and EC ₅₀value (Fig. 7).EPO-DDD2 and 734-EPO is shown as the Bioactivity of the rhEPO of about 10%.

Embodiment 21. is based on the generation of the DDD module of G-CSF (G-CSF)

For the structure of G-CSF-DDD2-pdHL2 of expressing in mammalian cell

By the cDNA sequence of pcr amplification G-CSF, produce the G-CSF merging the polypeptide be extremely made up of SEQIDNO:88 at its C-terminal.Total length human G-CSF cDNA clone (InvitrogenIMAGE people cat#97002RGCloneID5759022) is used to carry out pcr amplification as template and following oligonucleotide as primer:

g-CSFXbaI is left

TCTAGACACAGGACCTCATCATGGCTGGACCTGCCACCCAG(SEQIDNO:93)

g-CSFBamHI-is right

GGATCCATGATGGTGATGATGGTGTGACTTGGGCTGGGCAAGGTGGCGTAG(SEQIDNO:94).

Pcr amplification primer is cloned into pGemT carrier.Preparation DDD2-pdHL2 mammalian expression vector, by being connected it with G-CSF with the digestion of XbaI with BamHI restriction endonuclease.Use XbaI and BamHI to cut G-CSF amplimer from pGemT, connect into DDD2-pdHL2 carrier to produce expression vector G-CSF-DDD2-pdHL2.

The mammalian cell expression of G-CSF-DDD2

G-CSF-pdHL2 SalI enzymic digestion and linearized, is then stably transfected into Sp/ESF myeloma cell by electroporation method by it.Clone is selected with the substratum containing 0.15 μM of MTX.The G-CSF-DDD2 of about 0.15mg/L is produced by sandwich ELISA display clone #4.

From the batch culture purifying G-CSF-DDD2 cultivated rolling bottle

Clone #4 is equipped with in the rolling bottle of hybridoma SFM and the 0.4 μM MTX of 20L altogether at 34 and increases, reached end eventually and cultivate.By centrifugal clarification supernatant liquor, carry out filtering (0.2 μM), dialyse to 1X binding buffer liquid (10mM imidazoles, 0.5MNaCl, 50mMNaH ₂pO ₄, pH7.5) in, concentrated.By IMAC purified concentration thing.

The structure of G-CSF-DDD2 and the expression in intestinal bacteria

Also G-CSF-DDD2 is expressed as soluble protein by fermentable in intestinal bacteria.Use G-CSF-DDD2-pdHL2DNA as template, by pcr amplification encoding sequence.Use NdeI and XhoI restriction site that amplimer is cloned into pET26b coli expression carrier.By inducing LB shaking flask to carry out carrying out cell inner expression protein in BL21pLysS host cell in 12 hours with 100 μMs of IPTG at 18 DEG C.Utilize IMAC from cell lysate purification of soluble G-CSF-DDD2.

The structure of N-DDD2-G-CSF (C17S) and the expression in intestinal bacteria

Alternative G-CSF-DDD2 module is prepared, itself and the different halfcystine that do not match being to substitute with Serine on the 17th position of wild-type by merging DDD2 sequence and peptide transcribed spacer at the N-terminal of G-CSF (C17S).By N-DDD2-G-CSF (C17S) at expression in escherichia coli, IMAC is utilized to carry out purifying.

Embodiment 22.hR1-17S, comprises 4 and is connected to C _h3the generation of the DNL conjugate of N-DDD2-G-CSF (C17S) part of-AD2-IgG-hR1

After utilizing protein A affinity chromatography purifying, by under Redox Condition by C _h3-AD2-IgG-hR1 and excessive N-DDD2-G-CSF (C17S) combine to produce hR1-17S.The SE-HPLC of the hR1-17S of albumin A-purifying is analyzed to the main peak and shoulder (not shown) that show more macromolecule.The retention time of main peak is consistent with by IgG and 4 covalent complex that G-CSF group forms.Shoulder may owing to the non-covalent dimer of IgG-G-CSF conjugate.Under utilizing the DS-PAGE of Coomassie blue stain analysis and anti-G-CSF immunoblotting assay to be presented at non reducing conditions, described product has the Mr of the MW of the derivation corresponding to about 260kDa.Under the reducing conditions, the band (not shown) of 3 the composition polypeptide (N-DDD2-G-CSF (C17S), heavy chain-AD2 and light chain) representing hR1-17S is detected.

Embodiment 23. comprises generation and the purposes of the DNL construct of two different antibodies parts and cytokine

As mentioned above, 20-2b (by stopping-and the mono-specific immune cytokine that produces of-(DNL) method that locks (to comprise the four poly-IFN-α 2b being covalently attached to veltuzumab, humanization anti-CD 20 mAb)) in vitro and in human lymphoma heterograft, show extremely strong Anti-tumor active.But lymphoma and the leukemia of expressing or do not express CD20 are hardly expected to utilizing the treatment of 20-2b to have resistance.HLA-DR expresses on multiple hematopoetic tumor and some solid carcinoma.It may be able to be the therapeutical agent of more effective anti-extensive hematopoietic malignancies (comprise and express CD20, HLA-DR or both those tumours) by the dual specific immune cell factor of IFN-α target CD20 and HLA-DR.Due to each component (veltuzumab, the anti-HLA-DRF (ab) of multi-functional mixture ₂with IFN-α 2b) uniquely there is anti-tumor activity, we estimate that whether dual specific immune cell factor is potentially even than monospecific cytokine 20-2b more effective force.

We have reported generation and the qualification of the first dual specific MAb-IFN α of called after 20-C2-2b herein, and it is connected to IFN-α 2b and hL243 (the anti-HLA-DR of humanization of two copies of veltuzumab (humanization anti-CD 20) with comprising locus specificity; IMMU-114) stable F (ab) ₂.In vitro, 20-C2-2b suppresses each clone of 4 lymphomas and 8 myeloma cell lines, and except (a HLA-DR ^-/ CD20 ^-) in all the other strains beyond myelomatosis strain than monospecific CD20-target MAb-IFN α or to comprise the mixture of parental generation antibody and IFN α more effective, this shows that 20-C2-2b should be useful in the treatment of various hematopoietic malignancies.Anti-KMS12-BM (the CD20 that 20-C2-2b display is larger than the monospecific MAb-IFN α of target HLA-DR or CD20 ⁺/ HLA-DR ⁺myelomatosis) cytotoxicity, show that all 3 kinds of components of 20-C2-2b can cause cytotoxicity.Our discovery shows that the reactivity of given cell to MAb-IFN α depends on that it is to the susceptibility of IFN α and specific antibodies and the expression of antigen be targeted and density.

Because 20-C2-2b has antibody dependent cellular cytotoxicity (ADCC) but non-CDC, and can target CD20 and HLA-DR, so its treatment for the hemopoietic cancer of numerous any one or two kinds of antigens of expression is useful.Dual specific immune cell factor seems particularly useful in the elimination of the cancer stem cell eliminating the supposition relevant to myelomatosis (described cell have resistance to current treatment plan and express CD20 according to reports).

Materials and methods

antibody and cell culturesthe abbreviation used in following discussion is: 20 (C _h3-AD2-IgG-v-mab, anti-CD 20 IgGDNL module); C2 (C _h1-DDD2-Fab-hL243, anti-HLA-DRFab ₂dNL module); 2b (dimerization IFN α 2B-DDD2DNL module); 734 (anti-in-DTPAIgGDNL module, as non-targeted contrasts).Following MAb is provided by Immunomedics, Inc.: veltuzumab or v-mab (anti-CD 20 IgG ₁), hL243 γ 4p (Immu-114, anti-HLA-DRIgG ₄), mouse-anti-IFN α MAb and the anti-idiotype MAb of rat for v-mab (WR2) and hL243 (WT).The FBS of heat inactivation is available from Hyclone (Logan, UT).Every other cell culture medium and fill-in are purchased from InvitrogenLifeTechnologies (Carlsbad, CA).

Sp/ESF cell (having the clone derived from Sp2/0 of good development characteristic) is maintained in hybridoma serum-free medium.By NHL and MM cell cultures in the RPMI1640 substratum containing 10%FBS, 1mM Sodium.alpha.-ketopropionate, 10mML-glutamine and 25mMHEPES.Daudi, Ramos, Raji, Jeko-1, NCI-H929 and U266 human lymphoma cell system is purchased from ATCC (Manassas, VA).The source of MM clone is as follows: Dr.TakemiOtsuki (KawasakiMedicalSchool, Okayama, Japan) KMS11, KMS12-PE and KMS12-BM; Respectively from Dr.JoshuaEpstein (UniversityofArkansas, LittleRock, AK), Dr.KenjiOritani (OsakaUniversity, Osaka, and Dr.StevenRosen (NorthwesternUniversity Japan), Chicago, IL) CAG, OPM-6 and MM.1R.All cells system is all verified by supplier, obtains and go down to posterity to be no more than 50 times in 6 months of their uses.We do not verify described clone again.

dNL constructas in the previous embodiment, use DNL method, by IgG-AD2-module and DDD2-block combiner are produced monospecific MAb-IFN α (20-2b-2b, 734-2b-2b and C2-2b-2b) and dual specific HexAb (20-C2-C2).Four poly-IFN α 2b and MAbh734 [anti-indium-DTPAIgG will be comprised ₁] 734-2b-2b be used as non-targeted contrast MAb-IFN α.

The generation subsequently of the structure of mammalian expression vector and productivity clone and C _hthe purifying of 3-AD2-IgG-v-mab is disclosed in previous embodiment.Expressed recombination fusion protein has the C being connected to v-mab by the flexible connection body peptide of 15 amino acid longs _hthe AD2 peptide of the carboxyl terminal of 3 structural domains.The coexpression of heavy chain AD2 and light chain polypeptide causes the formation of the IgG structure being assembled with two AD2 peptides.By electroporation, expression vector is transfected into Sp/ESF cell (the genetic engineering modified clone of Sp2/0).PdHL2 carrier comprises the gene of tetrahydrofolate reductase, thus allows clonal selection and utilize methotrexate (MTX) to carry out gene amplification.From the clone of 96 orifice plate separating stables, the substratum containing 0.2 μM of MTX is utilized to select.By the C of sandwich ELISA screening and cloning _h3-AD2-IgG-vmab productive rate.Described module is produced in the rolling bottle with serum free medium.

As in embodiment 16 discuss, produce DDD-module, IFN α 2b-DDD2 by the carboxyl terminal merged with recombinating to people IFN α 2b through the flexible connection body peptide of 18 amino acid longs by DDD2 peptide.Also be that fusion rotein like this, expressed spontaneously forms stable homodimer for all DDD-modules.

As in embodiment 1-7 discuss carry out multiple C _hthe generation of 1-DDD2-Fab module, qualification and use.C is produced as follows from hL243-IgG-pdHL2 carrier _h1-DDD2-Fab-hL243 expression vector: cut C with SacII and EagI restriction enzyme _h1-hinge-C _h2-C _hthe sequence of 3 structural domains, is then replaced by the coding C cut from C-DDD2-hMN-14-pdHL2 expression vector with identical enzyme _hthe 507bp sequence of 1-DDD2.By electroporation by C _hafter 1-DDD2-Fab-hL243-pdHL2 is transfected into Sp/ESF cell, by using the productive rate of the clone screening anti-MTX with 96 hole microtiter plates of mouse Anti-Human κ chain bag quilt with the sandwich ELISA of prey fusion protein, the goat anti-human Fab that horseradish peroxidase is puted together in described fusion rotein use detects.Described module is produced in spinner culture thing.

Spinner culture thing in serum-free H-SFM substratum and batch feeding bio-reactor are produced and are caused the yield suitable with cytokine-DDD2 module with producing other other IgG-AD2 modules produced up to now.Respectively (the people such as Rossi as discussed previously, Blood2009,114:3864-71), MAbSelect (GEHealthcare) and His-SelectHF nickel affinity gel (Sigma) is used, by affinity chromatography purifying C from nutrient solution _h3-AD2-IgG-v-mab and IFN α 2b-DDD2.Will containing C _hthe nutrient solution of 1-DDD2-Fab-hL243 module is directly used in KappaSelect affinity gel (GE-Healthcare), is washed to baseline with PBS, then uses 0.1M glycine (pH2.5) to carry out wash-out.

SDS-PAGE and SE-HPLC is used to assess the purity (not shown) of DNL module.Analysis display under non reducing conditions, before DNL reaction, IFN α 2b-DDD2 and C _hthere is (not shown) in the dimeric forms that 1-DDD2-Fab-hL243 connects with disulfide linkage.This phenomenon always seen for DDD module is useful, because its protective reaction sulfydryl is from irreversible oxidation.Under comparing, C _hthe dimeric forms that 3-AD2-IgG-v-mab (not shown) is connected with disulfide linkage with monomer exists, and is reduced to monomer in DNL reaction.SE-HPLC analyzes consistent with irreducibility SDS-PAGE result, shows monomeric species and after reduction, is converted into the dimer module of monomeric form.Sulfydryl is protected in two kinds of forms by the disulfide linkage participated between AD2 cysteine residues.Reduction SDS-PAGE shows each module and is purified to close to homogeneous and the component polypeptides (not shown) being defined as comprising each module.For C _h3-AD2-IgG-v-mab, identifies heavy chain-AD2 and κ light chain.Resolve C _hhL243-Fd-DDD2 and the κ light chain polypeptide (not shown) of 1-DDD2-Fab-hL243.Resolved a master tape and a sub-band (not shown) of IFN α 2b-DDD2, described band is through determining to be respectively both the non-glycated and O-glycosylation species.

20-C2-2b is produced by DNLby 3 kinds of DNL module (C _h3-AD2-IgG-v-mab, C _h1-DDD2-Fab-hL243 and IFN-α 2b-DDD2) combine with equimolar amount to produce bsMAb-IFN α, 20-C2-2b.At room temperature carry out after stop step spends the night, adding Sleep-promoting factor B (2mM) to be conducive to disulfide formation (locking) under the reductive condition (1mM reduced glutathion) of gentleness.3 continuous affinity chromatography steps are used to be purified to 20-C2-2b close to homogeneous.At the beginning, utilize albumin A (MAbSelect) purifying DNL mixture, described albumin A is in conjunction with C _h3-AD2-IgG-v-mab group and eliminate unreacted IFN α 2b-DDD2 or C _h1-DDD2-Fab-hL243.His-SelectHF nickel affinity gel is used to be further purified by IMAC the material that albumin A combines, described affinity gel specific binding IFN α 2b-DDD2 part and eliminate any structure that there is not this group.Use the anti-idiotype affinity gel of hL243-, final treatment step can remove does not exist C _hany molecule of 1-DDD2-Fab-hL243.

It will be understood by those skilled in the art that the DNL mixture that affinitive layer purification can be used to comprise any combination of effector part, as long as the part of each of 3 kinds of effector parts can be obtained and be connected to column material.Selected DNL construct combines the DNL construct comprising each of 3 pillars of the part of each of 3 kinds of effect parts, and can in washing to remove after in conjunction with mixture by wash-out.

Following Examples represents several similar 20-C2-2b preparation.By the C of equimolar amount _h3-AD2-IgG-v-mab (15mg), C _h1-DDD2-Fab-hL243 (12mg) and IFN-α 2b-DDD2 (5mg) combines in 30-mL reaction volume, adds 1mM reduced glutathion in solution.After carrying out 16 hours under being room temperature, in mixture, add 2mM Sleep-promoting factor B, it is at room temperature kept other 6 hours.Reaction mixture is used for 5-mL albumin A affinity column, with PBS, this pillar is washed to baseline, then use 0.1M glycine (pH2.5) to carry out wash-out.Use 3MTris-HCl (pH8.6) neutralization containing the elutriant of 20mg protein of having an appointment, before for 5-mLHisSelectIMAC post, be dialyzed to HisSelect binding buffer liquid (10mM imidazoles, 300mMNaCl, 50mMNaH ₂pO ₄, pH8.0).With HisSelect binding buffer liquid, this pillar is washed to baseline, then use 250mM imidazoles, 150mMNaCl, 50mMNaH ₂pO ₄, pH8.0 carries out wash-out.

IMAC elutriant containing 11.5mg protein of having an appointment is directly used in WP (anti-hL243) affinity column, with PBS, this pillar is washed to baseline, then use 0.1M glycine (pH2.5) to carry out wash-out.This process produces the highly purified 20-C2-2b of 7mg.This is about 44% of the theoretical yield of 20-C2-2b, is 50% of total parent material (in this example 16mg), produces byproduct 20-2b-2b and 20-C2-C2 of respective 25%.

analytical procedureuse AllianceHPLC system, utilize BioSuite250,4 μm of UHRSEC pillars (WatersCorp., MilfordMA) carry out size exclusion HPLC (SE-HPLC).By before the immunocomplex utilizing SE-HPLC analysis gained, immunoreactivity is assessed in excessive WT, anti-IFN α or WR2 and 20-C2-2b mixing.Under reduction and non reducing conditions, use 12% and 4-20% gradient Tris-glycine gels (Invitrogen, Gaithersburg, MD) carry out SDS-PAGE respectively.

Use and carry out electro-spray ionization flight time (ESI-TOF) liquid chromatography/mass spectrometry (LC/MS) with the 1200-series HPLC of 6210TOFMS (AgilentTechnologies, SantaClara, CA) coupling.With 10mMtris (2-propyloic) phosphine reductase 12 0-C2-2b at 60 DEG C, carry out 30 minutes, then Poroshell300SB is utilized, 5 μm of C8 pillars (Agilent), use the acetonitrile gradient in 0.1% water-containing formic acid of the 20-90% of 10-minute, resolved by reversed-phase HPLC (RP-HPLC).For TOFMS, kapillary and disintegrator voltage (fragmentorvoltage) are respectively provided to 5500 and 200V.

ILiteHumanInterferonAlphaCell-BasedAssay test kit (PBLInterferonSource, Piscataway, NJ) is used to measure the specific activity of IFN α 2b.Peg-interferon α-2b (ScheringCorp) is as positive control.

cell binding and apoptosisthe scheme (Millipore, Billerica, MA) being respectively used to GuavaExpress and GuavaNexin using GuavaPCA and reagent, software and advise, by flow cytometry assessment Cell binding and apoptosis.Combination is measured, by viable cell at 4 DEG C with to be diluted in together with MAb or the MAb-IFN α in 1%BSA-PBS incubation 1 hour.Sedimentation cell, the mouse-anti-human igg-Fc (SouthernBiotech, Birmingham, AL) being to put together with 2 μ g/mL at 4 DEG C PE, together before incubation 1 hour, washs described cell with 1%BSA-PBS.After 3 washings, measured by flow cytometry and combine.Apoptosis is measured, before quantitative annexin-V-positive cell %, by cell (5 × 10 ⁵/ mL) incubation 48 hours together with MAb or the MAb-IFN α specified is in 24 orifice plates.

vitro cytotoxicityconcentration ever-increasing appointment reagent deposit in case by cell with predetermined best initial density (1-2.5 × 10 ⁵individual cell/mL) be seeded in 48 orifice plates (300 μ L/ hole), at 37 DEG C, carry out incubation, until the density of untreated cell increases to more than 10 times (4-7 days).Relative viable cell density at the end of using CellTiter96 Cell Proliferation assay (Promega, Madison, WI) to determine to measure.

daudi is consumed in vitro from whole bloodblood sample is collected under the scheme ratified by New England institutional review board (Wellesley, MA).By Daudi (5 × 10 ⁴) cell mixes with the Heparinised whole blood (150L) from healthy volunteer, by MAb or the MAb-IFN α of itself and 1nM at 37 DEG C and 5%CO ₂under incubation 2 days together.Cell FITC-anti-CD19, FITC-anti-CD14, anti-CD3 or the APC-mouse IgG of APC- ₁isotype controls MAb (BDBiosciences, SanJose, CA) dyes, and then uses FACSCalibur (BDBiosciences) to be analyzed by flow cytometry.Daudi cell is CD19+ and is in monocyte gate.Normal B and T cell are respectively CD19+ and CD3+ cell, are in monocyte gate.Monocyte is the CD14+ cell be in monocyte gate.

Result

the generation of 20-C2-2b and qualificationby by IgG-AD2 module, C _hthe dimerization DDD-module C that 3-AD2-IgG-v-mab is different from two kinds _h1-DDD2-Fab-hL243 and IFN α 2b-DDD2 combines and produces dual specific MAb-IFN α.Due to the random incorporation of arbitrary DDD-module and two AD2 groups, expect except 20-C2-2b, also form two kinds of byproduct 20-C2-C2 and 20-2b-2b.

20-C2-2b (~ 305kDa) is resolved to the band between band that cluster is positioned at 20-C2-C2 (~ 365kDa) and 20-2b-2b (255kDa) by non-reduced SDS-PAGE (not shown).Reduction SDS-PAGE parses 5 peptide species (v-mabHC-AD2, hL243Fd-DDD2, IFN α 2b-DDD2 and altogether migration v-mab and the hL243 κ light chain) (not shown) comprising 20-C2-2b.IFN α 2b-DDD2 and hL243Fd-DDD2 is not present in 20-C2-C2 and 20-2b-2b.MAbSelect is combined in all 3 kinds of main species produced in DNL reaction, but eliminates any excessive IFN α 2b-DDD2 and C _h1-DDD2-Fab-hL243.His-Select mainly comprises 20-C2-C2 (not shown) in conjunction with fraction.20-2b-2b (not shown) is comprised in conjunction with fraction from WT affinity chromatography.By each sample experience SE-HPLC and immunoreactivity analysis, this revises result and the conclusion of SDS-PAGE analysis.

After reductase 12 0-C2-2b, resolve its 5 kinds composition polypeptide by RP-HPLC, each peak produces single ESI-TOF overlap and closes mass spectrum (deconvolutedmassspectra) (not shown).Natural but not the restructuring IFN α 2 of bacterial expression on Thr-106 by O-glycosylation (people such as Adolf, BiochemJ1991; 276 (Pt2): 511-8).We determine that the polypeptide comprising IFN α 2b-DDD2 module of about 15% is by O-glycosylation, and resolve (not shown) by RP-HPLC and SDS-PAGE by non-glycosylated polypeptide.The LC/MS of 20-C2-2b is analyzed and identifies the O-glycosylation of IFN α 2b-DDD2 and non-glycosylated kind (not shown) with the Mass accuracy of 15ppm and 2ppm respectively.The glycan that the quality display O-of the O-glycoforms observed connects has structure NeuGc-NeuGc-Gal-GalNAc, also predicts this point (not shown) for 20-2b-2b (<1ppm).V-mab and hL243 κ chain and hL243-Fd-DDD2 (not shown) are accredited as the kind of single unmodified by LC/MS, observed quality matches quality as calculated (<35ppm).Two kinds of main sugar forms of v-mabHC-AD2 are accredited as has 53,714.73 (70%) and 53, the quality of 877.33 (30%), represents G0F and G1FN-glycan respectively, described glycan usually with IgG in conjunction with (not shown).Analyze and also confirm that the aminoterminal of HC-AD2 is modified to Pyrrolidonecarboxylic acid, as have N-terminal glutamine polypeptide predicted.

The SE-HPLC analytical solution of 20-C2-2b is separated out and its uniform quality as calculated and the major protein peak of retention time (6.7 minutes) between larger 20-C2-C2 (6.6 minutes) and the quality of less 20-2b-2b (6.85 minutes), and some oneself that may represent through IFN α 2b is associated the higher molecular weight peaks (not shown) of the non-covalent dimer of formation.

Immunoreactivity measures the uniformity (not shown) of display 20-C2-2b (each molecule comprises 3 kinds of functional groups).The incubation of 20-C2-2b and the excessive antibody of any one for three kinds of ingredients causes the quantitative formation of high molecular immunocomplex and the disappearance at 20-C2-2b peak.His-Select and WT is affine does not react (not shown) with WT and anti-IFN alpha immunization respectively in conjunction with fraction.

cell bindingmAb-IFN α shows the binding affinity (Fig. 8 A) similar to their parental generation MAb.In sub-saturated concentration, 20-C2-2b with hL243 γ 4p is observed similar in conjunction with level.The antigen density of HLA-DR is about 6 times of the CD20 in these cells, thus allows the combination of more 20-C2-2b compared with 20-2b-2b.In conjunction with the binding curve of nonlinear regression model (NLRM) analysis, applying unit point proves that 20-C2-2b can to obtain compared with 20-2b-2b the B of comparatively 4 times _max, between their binding affinity, do not observe significant difference (K _dabout 4nM) (Fig. 8 B).

the biological activity of IFN αthe reporter gene assay based on cell is used to measure the specific activity of multiple MAb-IFN α, by it compared with peg-interferon α-2b (Fig. 8 C).As expected, there is the remarkable specific activity lower than 20-2b-2b (4447IU/pmol) or 734-2b-2b (3764IU/pmol) of specific activity (2454IU/pmol) of the 20-C2-2b of two IFN α 2b groups, then still higher than peg-interferon α-2b (P<0.001).Difference between 20-2b-2b and 734-2b-2b is not remarkable.When IU/pmol stdn for total IFN α, the specific activity change between all reagent is minimum.Based on these data, the specific activity of each IFN α 2b group of MAb-IFN α is about 30% of restructuring IFN α 2b (~ 4000IU/pmol).

vitro cytotoxicity: NHLthe result of the vitro cytotoxicity of B cell NHL is used to be summarized in table 3.Report the relative antigen density (people such as Stein, Blood2010, at press) of HLA-DR with CD20 of each clone.Target index (targetingindex) (TI) represents that the multiple of the effect of target MAb-IFN α increases, by EC compared with non-targeted MAb-IFN α (734-2b-2b) ₅₀value is converted into total IFN α concentration (I-EC ₅₀).The cell killing that Daudi causes IFN α 2 is very responsive, as utilized non-targeted MAb-IFN α, 734-2b-2b (I-EC ₅₀=14pM) prove.With monospecific 20-2b-2b (I-EC ₅₀=0.4pM) CD20 on target Daudi makes effect be enhanced to 25 times (TI=25), with previous result (people such as Rossi, Blood2009 further; 114:3864-71) conform to.Dual specific 20-C2-2b (I-EC ₅₀=0.08pM; TI=125) effect is enhanced to 5 times of 20-2b-2b, and this is attributable to antigen density and its high affinity tetravalence tumour combination possibly of the increase of HLA-DR.The intracellular signaling of hL243-induction unlikely causes extra cytotoxicity on these lower concentrations.The mixture of v-mab, hL243 γ 4p and 734-2b-2b (v-mab+hL243+734-2b) is equal to independent 734-2b-2b, and this intracellular signaling supporting that conclusion: hL243-induces does not cause the high TI of 20-C2-2b.

Only induce the apoptosis (Fig. 9 A) of Daudi without v-mab or the hL243 γ 4p of 10pM with any MAb-IFN α of 1pM.The process of 20-2b-2b or 20-C2-2b is utilized to produce Apoptosis (P<0.0005) more significant than 734-2b-2b or v-mab+hL243+734-2b.Significant difference is not observed between 734-2b-2b and described mixture.

734-2b-2b is to Raji (I-EC ₅₀=32nM) and Ramos (I-EC ₅₀>80nM) impact is not too large, produces the maximum suppression (I of only 62% and 35% respectively _max).Under these conditions, hL243 γ 4p but not v-mab (not shown) suppresses these Burkitt lymphoma strains.For Raji, the enhancing of the effect of the 20-C2-2b (TI=118) observed is more than 50 times of 20-2b-2b (TI=2), and the HLA-DR antigen density of described Raji is more much bigger than CD20 density.Different from Daudi and Raji, the density of HLA-DR with CD-20 on Ramos is similar, but the TI of 20-C2-2b is 15 times of 20-2b-2b, shows the cumulated activity of hL243 and IFN α 2b.

For the single agents more effective force that Raji and Ramos, v-mab+hL234+734-2b mixture is more independent than any one.Target IFN α 2b is vital for acquisition maximum effectiveness.In each strain of 3 kinds of Burkitt lymphoma strains, 20-C2-2b is more effective than v-mab+hL234+734-2b, and described v-mab+hL234+734-2b comprises the anti-CD 20 of similar number and the IFN α 2b of anti-HLA-DRFab and doubling dose (and activity).

Mantle cell lymphoma Jeko-1 works as strong (EC to the reacting phase of hL243 γ 4p ₅₀=0.4nM), but to IFN α 2b not too responsive (for 734-2b-2b, effect is minimum).Any process comprising hL243 is better than 20-2b-2b (EC ₅₀=1nM).20-C2-2b and hL243 γ 4p or v-mab+hL243+734-2b display of comparing is enhanced to the effect of 2 times.On 0.5nM, hL243 γ 4p and 734-2b-2b induces the apoptosis of similar level and their effect is obviously accumulation, because utilize the process of v-mab+hL243+734-2b to cause the annexin-V-positive cell (Fig. 9 A) with arbitrary independent Reagent evaluation more about 2 times of quantity.Can suppose, v-mab mixture almost without contribution because it only has faint effect individually.20-C2-2b and mixture are better than 20-2b-2b (P<0.002) because of the effect of hL243.

vitro cytotoxicity: myelomatosisalthough 8 MM clone HLA-DR level (and only having KMS12-BM to express CD20) upper with to the susceptibility of IFN α 2b different (FIG.10), all 20-C2-2b is reacted.The dose-response curve of each of 8 MM clones testing is shown in Figure 11, and result is summarized in table 3.Such as, 5 kinds is that highly reaction is (for 734-2b-2b, I-EC to IFN α 2 ₅₀but HLA-DR antigen density is different <1nM).Certainly, the CAG only with high HLA-DR density is suppressed (>1nM) by hL243 γ 4p, and in higher concentration, show enhancing (increase) is to the reaction of the mixture (hL243+734-2b) of hL243 γ 4p and 734-2b-2b.20-C2-2b (I-EC ₅₀=10pM) for the remarkable effect (TI=55) strengthened of CAG display.

With 1nM but not with 0.1 or 0.01nM hL243 γ 4p, 20-2b-2b, 734-2b-2b or hL243+734-2b process after, the apoptosis of CAG is clearly (Fig. 9 B).The even 20-C2-2b cell death inducing of 0.01nM, and level is observed for 0.1nM20-C2-2b process the higher of the on level terms or ratio produced with by any other of 10 times of (1nM) concentration.

The effect of the enhancing of 20-C2-2b is significant, but lower for OPM6 (TI=2), U266 (TI=7) and MM.1R (TI=10) (separately for height-IFN α-reactive but have lower HLA-DR density and do not suppressed by hL243 γ 4p) effect.Do not observe the effect of 20-C2-2b to the enhancing of NCI-H929 (it is height IFN α-reactive but is HLA-DR).

KMS12-BM has high HLA-DR and CD20 antigen density, and surprisingly, by 20-2b-2b (I-EC ₅₀=31nM) but not 734-2b-2b (I-EC ₅₀>100nM) or v-mab suppress.KMS12-BM is to v-mab+hL243+734-2b (EC ₅₀=3nM) reaction with to hL243+734-2b (EC ₅₀=0.7nM) reacting phase stronger, thus be better than independent hL243 γ 4p (EC ₅₀=3.5nM).Each process of these process causes apoptotic strong induction, and relative level conforms to (Fig. 9 C) with vitro cytotoxicity result.In addition, v-mab+hL243 induces more apoptosis compared with independent hL243 γ 4p, but the apoptosis that induction is less compared with v-mab+hL243+734-2b.These results show for HLA-DR ⁺/ CD20 ⁺mM cell, 20-C2-2b (EC ₅₀=0.1nM) the activity of all 3 kinds of components can cause cytotoxicity upon combination, even if in fact two in them do not have effect when used alone.

The DNL construct that assessment two is extra in KMS12-BM helps the effect illustrating the enhancing of 20-C2-2b.Comprise hL243IgG ₁cytotoxicity (the EC not too strong compared with 20-C2-2b with MAb-IFN α (called after C2-2b-2b) display of four poly-IFN α 2b (twice for the IFN α 2b of 20-C2-2b) ₅₀=0.4nM) and faint apoptosis-inducing, thus support the contribution of v-mab.Further announcement be find: the dual specific MAb of 20-C2-C2 (comprising v-mab and 4 HLA-DRFab) shows high-caliber apoptosis-inducing and is enhanced to the effect (EC of more than 50 times with hL243 γ 4p compared with ₅₀=0.06nM), showing the HLA-DR that exists and CD20 and 20-C2-2b, be cross-linked can by the signal transduction cascade of uniqueness inducing cytotoxic effectively.Although each construct is strong (EC ₅₀but 20-C2-C2 (I <0.5nM), _max=67%) and C2-2b-2b (I _max=70%) not as 20-C2-2b (I _max=99%) equally effectively kill and wound KMS12-BM, thus support to need all three kinds of components to obtain maximum effect.V-mab+hL243+734-2b (I _max=87%) mixture is onlyly cause the process killed and wounded more than 70% to support this hypothesis.

Integrate, these digital proof antigen densities and the susceptibility to the effect of IFN α 2b and the effect of target MAb are all the important determinative of specific cells system to the vitro reactions of different MAb-IFN α.But, Vitro Tumor kill and wound by ADCC and immune effector cell be used for strengthen, described effector cell can be stimulated by the IFN α 2b of high local concentrations.

effector function in human serum and stabilitywe had previously reported 20-2b-2b and shown the ADCC (people such as Rossi, the Blood2009 that strengthen compared with its parental generation v-mab; 114:3864-71).By design, hL243 γ 4p has the ADCC (people such as Stein, the Blood2006 that weaken; 108:2736-44).But 20-C2-C2 and v-mab compares and induces the ADCC (not shown) that significantly (P=0.0091) is stronger.Notably, 20-C2-2b and 20-2b-2b (P=0.0040) or 20-C2-C2 (P=0.0115) compares and induces significantly stronger ADCC, shows that IFN α 2b's exists reinforcing effect subfunction.As previous for 20-2b-2b prove (people such as Rossi, Blood2009; 114:3864-71), 20-C2-2b does not induce CDC (not shown) in vitro.

Two kinds for measuring the stability of 20-C2-2b in human serum different assay methods provide closely similar result, and the loss in display about 3.5%/sky, carries out 11 days remaining about 65% (not shown)s afterwards at 37 DEG C.

nHL is consumed in vitro from people's whole bloodas shown in Figure 12, compared with 20-2b-2b (69%), v-mab (49% consumes), hL243 γ 4p (46%) or 734-2b-2b (10%), 20-C2-2b (91%) more effectively consumes Daudi cell from whole blood (in vitro).Two kinds of target MAb-IFN α are less to the toxicity of the toxicity ratio of normal B cells to Daudi.Under these conditions, B cell is got rid of in a large number by 20-C2-2b (57%) and hL243 γ 4p (41%), but is not got rid of by v-mab, 734-2b or 20-2b-2b.Monocyte is got rid of by hL243 γ 4p (48%), 734-2b-2b (30%) and 20-2b-2b (21%), but is not got rid of by v-mab.20-C2-2b (98%) has high toxicity to monocyte.A kind of reagent is not had to have remarkable effect to T cell.Xue Shengshi t is utilized to check the statistical significance (P<0.001) determining the difference of each consumption % above specified.

Discuss

We and other researchist have reported and have comprised CD20-target MAb and the fusion rotein of IFN α and to compare with the combination of MAb with IFN α in xenotransplantation with homogenic type mouse model more effectively anti-NHL, this shows that MAb-IFN α can overcome the toxicity relevant to IFN α and Pk limits (people such as Rossi, Blood2009; 114:3864-71; The people such as Xuan, Blood2010; 115:2864-71).Although CD20 is the attracting candidate of the target MAb-IFN α therapy of B cell lymphoma, it expresses the malignant tumour being mainly defined in this pedigree, the low antigen density of some individual display.Herein, we have reported the first dual specific immune cell factor 20-C2-2b of selectively targeted CD20 and HLA-DR of IFN α 2b, thus extend the neoplastic hematologic disorder kind being suitable for this immune cell factor therapy potentially.

Anti-HLA-DRMAb cell death inducing (described apoptosis mediated by direct intracellular signaling and without the need to extra crosslinked) and be also effective inductor (people such as Stein, the Blood2006 of ADCC and CDC efficiently; 108:2736-44; The people such as Rech, LeukLymphoma2006; 47:2147-54).When ADCC can strengthen treatment potential, the pathogenesis of the side effect that CDC primary responsibility is relevant to MAb infusion (people such as vanderKolk, BrJHaematol2001; 115:807-11).By using human IgG ₄the constant region of isotype is carried out genetic engineering modified (to improve clinical safety) with the anti-HLA-DRMAb of the humanization compared (hL243 γ 4p) in this research, thus causes ADCC and CDC of reduction.20-C2-2b is unique in anti-HLA-DRMAb, because it is similar to hL243 γ 4p, there is not CDC, but has strong (enhancing) ADCC, and this makes this reagent become the attractive candidate of immunotherapy.

In ex vivo environment, the apoptosis that v-mab can be induced by intracellular signaling, ADCC and CDC consume cell.The intracellular signaling that MAb-IFN α can utilize ADCC and MAb-of enhancing and IFN α 2b-to induce, but can not CDC be utilized; And hL243-γ 4p is only limitted to direct signal conduction (people such as Stein, Blood2006; 108:2736-44).Although fail the full spectrum of activation of the IFN α-mediation obtaining the natural immunity and the adaptive immunity that may occur in vivo in this context, which provide pharmacokinetic data.The lymphoma cell ratio that 20-C2-2b consumes is higher to the consumption efficiency of normal B cells, and to T cell without effect.But it eliminates monocyte really efficiently.At v-mab in the adiaphorous situation of monocyte, after utilizing hL243 α 4p and MAb-IFN α process, observe consumption, 20-2b-2b and 734-2b-2b shows similar cytotoxicity.Therefore, the predictable more efficient of 20-C2-2b is owing to the compound action of anti-HLA-DR and IFN α, and this effect can be strengthened by HLA-DR target.These data show that monocyte consumption can be the pharmacokinetics effect relevant to anti-HLA-DR and IFN α therapy; But this side effect may be of short duration because monocyte population should from hemopoietic stem cell again live into.

We comprise the expression of HLA-DR and CD20 and the naturally occurring heterogeneity of antigen density and the reaction to the effect of IFN α, hL243 and v-mab at 4 NHL and 8 MM clones of research, and it all affects MAb-IFN alpha immunization therapy.6 and 8 clone (in 12 clones) suppressed (I to some extent by hL243 γ 4p and 734-2b-2b respectively _max>30%).20-C2-2b effectively suppresses (EC ₅₀<1nM) 11 clones in 12 clones, have EC for 5 clones ₅₀≤ 0.01nM.Even if the influenced minimum MM strain (KMS11) do not suppressed by 734-2b-2b also to respond (EC to 20-C2-2b ₅₀=17nM).

Except being HLA-DR ^-/ CD20 ^-nCI-H929 outside, in all cells system, observe the enhancing of 20-C2-2b relative to the effect of 734-2b-2b.Higher levels of HLA-DR antigen density and relevant to the TI of larger 20-C2-2b to the reaction of hL243, this demonstrate that the cumulated activity of IFN α and hL243 and the importance of target, even if in vitro.20-C2-2b is better than the mixture of v-mab+hL243+734-2b in 10 strains, highlights the impact of cancer target further, and this impact is much bigger in vivo, as previous for 20-2b-2b (people such as Rossi, the Blood2009 that prove; 114:3864-71).In addition, in body, target MAb-IFN α may cause virtuous Anti-tumor immune response.

Although the overwhelming majority bag celliferous primary MM sample right and wrong Clonal and there is plasma cell phenotype (CD138 ⁺/ CD19 ^-/ CD20 ^-), but the MM cancer stem cell of supposition is CD138 ^-and express B cell surface antigen, comprise CD45, CD19, CD20 and CD22 (people such as Matsui, Blood2004 that hint is memory B cell; 103:2332-6).Although various clinical method produces reaction, MM because being considered to the recurrence that mediated by cancer stem cell is still incurable substantially, and described MM has resistance to various therapy.Assuming that the B cell phenotype of stem cell facilitate the clinical study of the Rituximab carried out in MM.But, realize limited impact (people such as Treon, the JImmunother2002 on result; 25:72-81).

In vitro results about KMS12-BM is badly in need of, because it is similar to the MM stem cell mentioned, is CD20 ⁺.20-C2-2b shows the apoptosis of the KMS12-BM of strong cytotoxicity and powerful induction.Even if non-targeted MAb-IFN α and v-mab is invalid as single agents, but when combinationally using with hL243, they cause cytotoxicity all significantly.Described result also shows that the dual specific of CD20 and HLA-DR combines and enhancing further can be induced the cytotoxicity of these cells and them can be made extra (strong) signal of IFN α sensitivity.

The MAb-IFN α produced by DNL shows the activity suitable with restructuring IFN α.Recently, the people such as Xuan have reported IFN alpha active (people such as Xuan, the Blood2010 that the anti-CD 20-IFN alpha fusion protein display prepared by conventional recombination engineering is decreased to 1/300; 115:2864-71).This similar Daudi xenotransplantation research relatively in be noticeable, the wherein 20-2b-2b of single 17ng dosage and dosage (more than 5000 times) (people such as Xuan, the Blood2010 for 3 30 μ g of the anti-CD 20-hIFN α that recombinates; 115:2864-71) compare and significantly improve survival rate (people such as Rossi, Blood2009; 114:3864-71).The research of the tumour of secretion IFN α is used to show immune response (people such as Ferrantini, the Biochimie2007 of the enhancing caused by the IFN α locally concentrated; 89:884-93).When this also may utilize high reactivity MAb-IFN α to obtain, the activity of the reduction of conventional restructuring MAb-IFN α effectively may not be recruited and stimulate Anti-tumor immune response, as by the people such as Xuan the (Blood2010 that reports; 115:2864-71).

Dual specific MAb-IFN α 20-C2-2b is attracting in treatment NHL, because each composition of 3 compositions this disease anti-actively.This research display 20-C2-2b is also useful for the treatment of MM and other hematopoietic malignancies.

It will be understood by those skilled in the art that and can use described herein together with using the method for dual specific immune factor or other DNL construct (comprising 3 kinds of different effect thing parts) and any combination of the antibody of DNL construct, antibody fragment, cytokine or other effector can be integrated in order to produce.

***

According to present disclosure preparation and disclosed herein and claimed all compositions and method can be used and without the need to undo experimentation.Although describe composition and method by preferred embodiment, it should be apparent that for those skilled in the art and can change the order of composition and method or the method described in this article or step and not deviate from concept of the present invention, spirit and scope.More specifically, some reagent of chemistry and physiology being all correlated with can be used for alternative reagent described herein and can obtain same or analogous result.It should be apparent that all this type of similar to substitute and modification is considered to such as by claims in determined spirit of the present invention, scope and concept for those skilled in the art.

Claims (25)

1. one kind comprises DNL (stop and the lock) construct of 3 kinds of different effect thing parts, wherein said effector part is connected to two DDD from protein kinase A (PKA) (dimerization and dockerin domain) part and the AD from AKAP albumen (anchoring domain) part, and wherein said two DDD part forms dimer and combines to form DNL construct with AD part.

2. DNL construct according to claim 1, wherein said DDD part has the aminoacid sequence from people RI α, RI β, RII α or RII β PKA.

3. DNL construct according to claim 1, wherein said effector part comprises the cytokine of first antibody or antibody fragment, second antibody or antibody fragment and one or more copy.

4. DNL construct according to claim 3, wherein said first antibody or antibody fragment and second antibody or antibody fragment are in conjunction with two kinds of different antigens.

5. DNL construct according to claim 4, wherein said first and second antibody or antibody fragment conjugated antigen, described antigen is selected from carbonic anhydrase IX, CCCL19, CCCL21, CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, AFP, PSMA, CEACAM5, CEACAM-6, B7, the ED-B of fibronectin, factor H, FHL-1, Flt-3, folacin receptor, GROB, HMGB-1, hypoxia inducible factor (HIF), HM1.24, insulin-like growth factor-i (ILGF-1), IFN-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-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5, PAM4 antigen, NCA-95, NCA-90, Ia, HM1.24, EGP-1, EGP-2, HLA-DR, tenascin, Le (y), RANTES, T101, TAC, Tn antigen, Thomson-Friedenreich antigen, tumor necrosis antigens, TNF-α, TRAIL acceptor (R1 and R2), VEGFR, EGFR, PlGF, complement factor C_3, C3a, C3b, C5a, C5 and oncoprotein.

6. DNL construct according to claim 5, wherein said first and second antibody or antibody fragment are selected from hR1 (anti-IGF-1R), hPAM4 (anti-Saliva Orthana), hA20 (anti-CD 20), hA19 (anti-CD19), hIMMU31 (anti-AFP), hLL1 (anti-CD74), hLL2 (anti-CD22), hMu-9 (anti-CSAp), hL243 (anti-HLA-DR), hMN-14 (anti-CEACAM5), hMN-15 (anti-CEACAM6), hRS7 (anti-EGP-1) and hMN-3 (anti-CEACAM6).

7. DNL construct according to claim 4, wherein said cytokine is selected from people MIF (macrophage migration inhibiting factor), HMGB-1 (high mobility group frame albumen 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-23, IL-24, CCL19, CCL21, IL-8, MCP-1, RANTES, MIP-1A, MIP-1B, ENA-78, MCP-1, IP-10, Gro-, eotaxin, interferon-' alpha ',-β,-λ, G-CSF, GM-CSF, SCF, PDGF, MSF, Flt-3 part, erythropoietin, thrombopoietin, CNTF, leptin, oncostatin M, VEGF, EGF, FGF, PlGF, Regular Insulin, hGH, thyrocalcitonin, Factor IX, IGF, somatostatin, tissue plasminogen activator and LIF.

8. DNL construct according to claim 4, wherein said first antibody or antibody fragment are veltuzumab, and described second antibody or antibody fragment are hL243, and described cytokine is human interferon-alpha 2b.

9. DNL construct according to claim 8, wherein said hL243 antibody or antibody fragment comprise heavy CDR sequences CDR1 (NYGMN, SEQIDNO:1), CDR2 (WINTYTREPTYADDFKG, and CDR3 (DITAVVPTGFDY SEQIDNO:2), and CDR sequence CDR1 (RASENIYSNLA SEQIDNO:3), SEQIDNO:4), CDR2 (AASNLAD, and CDR3 (QHFWTTPWA, SEQIDNO:6) SEQIDNO:5).

10. DNL construct according to claim 1, wherein said 3 kinds of effector parts are fusion roteins, and each fusion rotein comprises AD or DDD part.

11. DNL constructs according to claim 1, wherein said effector part is selected from protein, peptide, antibody, antigen binding antibody, immunomodulator, cytokine, hormone, enzyme, antisense oligonucleotide, siRNA, toxin, rnase, heterologous antigen, polyoxyethylene glycol (PEG), anti-angiogenic agent, cytotoxic agent and short apoptosis agent.

12. DNL constructs according to claim 1, wherein said DDD part has aminoacid sequence, front 44 amino acid of SEQIDNO:20, front 44 amino acid of SEQIDNO:21, SEQIDNO:22 or SEQIDNO:59 of SEQIDNO:13, SEQIDNO:14, SEQIDNO:17, SEQIDNO:18.

13. DNL constructs according to claim 1, wherein said AD part has SEQIDNO:15, SEQIDNO:16, SEQIDNO:19, SEQIDNO:23, SEQIDNO:24, SEQIDNO:25, SEQIDNO:26, SEQIDNO:27, SEQIDNO:28, SEQIDNO:29, SEQIDNO:30, SEQIDNO:31, SEQIDNO:32, SEQIDNO:33, SEQIDNO:34, SEQIDNO:35, SEQIDNO:36, SEQIDNO:37, SEQIDNO:38, SEQIDNO:39, SEQIDNO:40, SEQIDNO:41, SEQIDNO:42, SEQIDNO:43, SEQIDNO:44, SEQIDNO:45, SEQIDNO:46, SEQIDNO:47, SEQIDNO:48, MSEQIDNO:49, SEQIDNO:50, SEQIDNO:51, SEQIDNO:52, SEQIDNO:53, SEQIDNO:54, SEQIDNO:55, SEQIDNO:56, the aminoacid sequence of SEQIDNO:57 or SEQIDNO:58.

14. 1 kinds, to the method for experimenter's administer cytokines, comprise and use DNL mixture according to claim 4 to experimenter.

15. methods according to claim 14, wherein said first and second antibody or antibody fragment conjugated antigen, described antigen is selected from carbonic anhydrase IX, CCCL19, CCCL21, CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, AFP, PSMA, CEACAM5, CEACAM-6, B7, the ED-B of fibronectin, factor H, FHL-1, Flt-3, folacin receptor, GROB, HMGB-1, hypoxia inducible factor (HIF), HM1.24, insulin-like growth factor-i (ILGF-1), IFN-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-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5, PAM4 antigen, NCA-95, NCA-90, Ia, HM1.24, EGP-1, EGP-2, HLA-DR, tenascin, Le (y), RANTES, T101, TAC, Tn antigen, Thomson-Friedenreich antigen, tumor necrosis antigens, TNF-α, TRAIL acceptor (R1 and R2), VEGFR, EGFR, PlGF, complement factor C_3, C3a, C3b, C5a, C5 and oncoprotein.

16. methods according to claim 15, wherein said first and second antibody or antibody fragment are selected from hR1 (anti-IGF-1R) hPAM4 (anti-Saliva Orthana), hA20 (anti-CD 20), hA19 (anti-CD19), hIMMU31 (anti-AFP), hLL1 (anti-CD74), hLL2 (anti-CD22), hMu-9 (anti-CSAp), hL243 (anti-HLA-DR), hMN-14 (anti-CEA), hMN-15 (anti-CEA), hRS7 (anti-EGP-1) and hMN-3 (anti-CEA).

17. methods according to claim 14, wherein said cytokine is selected from people MIF (macrophage migration inhibiting factor), HMGB-1 (high mobility group frame albumen 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-23, IL-24, CCL19, CCL21, IL-8, MCP-1, RANTES, MIP-1A, MIP-1B, ENA-78, MCP-1, IP-10, Gro-, eotaxin, interferon-' alpha ',-β,-λ, G-CSF, GM-CSF, SCF, PDGF, MSF, Flt-3 part, erythropoietin, thrombopoietin, CNTF, leptin, oncostatin M, VEGF, EGF, FGF, PlGF, Regular Insulin, hGH, thyrocalcitonin, Factor IX, IGF, somatostatin, tissue plasminogen activator and LIF.

18. methods according to claim 14, wherein said first antibody or antibody fragment are veltuzumab, and described second antibody or antibody fragment are hL243, and described cytokine is human interferon-alpha 2b.

19. 1 kinds of treatments are selected from the method for the disease of cancer, immune dysfunction and autoimmune disorder, comprise and use DNL construct according to claim 1 to the experimenter suffering from described disease.

20. methods according to claim 19, wherein said cancer is selected from non_hodgkin lymphoma, B cell lymphoma, B cell leukemia, t cell lymphoma, T cell leukemia, acute lymphocytic leukemia, chronic lymphoid leukemia, Burkitt lymphoma, He Jiejin lymphomas, hairy cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, multiple myeloma, neurospongioma, Walden Si Telun macroglobulinemia, cancer, melanoma, sarcoma, neurospongioma, skin carcinoma, oral carcinoma, gastrointestinal cancer, lung road cancer, lung cancer, mammary cancer, ovarian cancer, prostate cancer, uterus carcinoma, endometrial cancer, cervical cancer, bladder cancer, carcinoma of the pancreas, osteocarcinoma, liver cancer, carcinoma of gallbladder, kidney and carcinoma of testis.

21. methods according to claim 19, wherein said autoimmune disorder is selected from acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematous, systemic lupus erythematosus, rheumatic fever, polyglandular syndrome, bullous pemphigoid, diabetes, purpura,Henoch-Schonlein, ephritis after streptococcal infection, erythema nodosum, Gao An (family name) arteritis, Addison disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangiitis obliterans, xerodermosteosis, primary biliary cirrhosis, struma lymphomatosa, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris, Wei Genashi granuloma, membranous nephropathy, amyotrophic lateral sclerosis, myelophthisis, giant cell arteritis/polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriatic or fibrosing alveolitis.

22. methods according to claim 19, wherein said first and second antibody or antibody fragment conjugated antigen, described antigen is selected from carbonic anhydrase IX, CCCL19, CCCL21, CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, AFP, PSMA, CEACAM5, CEACAM-6, B7, the ED-B of fibronectin, factor H, FHL-1, Flt-3, folacin receptor, GROB, HMGB-1, hypoxia inducible factor (HIF), HM1.24, insulin-like growth factor-i (ILGF-1), IFN-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-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5, PAM4 antigen, NCA-95, NCA-90, Ia, HM1.24, EGP-1, EGP-2, HLA-DR, tenascin, Le (y), RANTES, T101, TAC, Tn antigen, Thomson-Friedenreich antigen, tumor necrosis antigens, TNF-α, TRAIL acceptor (R1 and R2), VEGFR, EGFR, PlGF, complement factor C_3, C3a, C3b, C5a, C5 and oncoprotein.

23. methods according to claim 19, wherein said first and second antibody or antibody fragment are selected from hR1 (anti-IGF-1R) hPAM4 (anti-Saliva Orthana), hA20 (anti-CD 20), hA19 (anti-CD19), hIMMU31 (anti-AFP), hLL1 (anti-CD74), hLL2 (anti-CD22), hMu-9 (anti-CSAp), hL243 (anti-HLA-DR), hMN-14 (anti-CEA), hMN-15 (anti-CEA), hRS7 (anti-EGP-1) and hMN-3 (anti-CEA).

24. methods according to claim 19, wherein said cytokine is selected from people MIF (macrophage migration inhibiting factor), HMGB-1 (high mobility group frame albumen 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-23, IL-24, CCL19, CCL21, IL-8, MCP-1, RANTES, MIP-1A, MIP-1B, ENA-78, MCP-1, IP-10, Gro-, eotaxin, interferon-' alpha ',-β,-λ, G-CSF, GM-CSF, SCF, PDGF, MSF, Flt-3 part, erythropoietin, thrombopoietin, CNTF, leptin, oncostatin M, VEGF, EGF, FGF, PlGF, Regular Insulin, hGH, thyrocalcitonin, Factor IX, IGF, somatostatin, tissue plasminogen activator and LIF.

25. methods according to claim 19, wherein first antibody or antibody fragment are veltuzumab, and described second antibody or antibody fragment are hL243, and described cytokine is human interferon-alpha 2b.