CN102596235A - Compositions and methods of use of immunotoxins comprising ranpirnase (RAP) show potent cytotoxic activity - Google Patents

Compositions and methods of use of immunotoxins comprising ranpirnase (RAP) show potent cytotoxic activity Download PDF

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CN102596235A
CN102596235A CN2010800386149A CN201080038614A CN102596235A CN 102596235 A CN102596235 A CN 102596235A CN 2010800386149 A CN2010800386149 A CN 2010800386149A CN 201080038614 A CN201080038614 A CN 201080038614A CN 102596235 A CN102596235 A CN 102596235A
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antibody
seq
cancer
rap
cell
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C-H·张
D·M·戈登堡
E·A·罗斯
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Immunomedics Inc
IBC Pharmaceuticals Inc
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Immunomedics Inc
IBC Pharmaceuticals Inc
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Priority claimed from US12/644,146 external-priority patent/US7981398B2/en
Priority claimed from US12/731,781 external-priority patent/US8003111B2/en
Priority claimed from US12/752,649 external-priority patent/US8034352B2/en
Priority claimed from US12/754,140 external-priority patent/US8722047B2/en
Priority claimed from US12/754,740 external-priority patent/US8562988B2/en
Application filed by Immunomedics Inc, IBC Pharmaceuticals Inc filed Critical Immunomedics Inc
Publication of CN102596235A publication Critical patent/CN102596235A/en
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Abstract

The present invention concerns methods and compositions for forming immunotoxin complexes having a high efficacy and low systemic toxicity. In preferred embodiments, the toxin moiety is a ranpirnase (Rap), such as Rap(Q). In more preferred embodiments, the immunotoxin is made using dock-and-lock (DNL) technology. The immunotoxin exhibits improved pharmacokinetics, with a longer serum half-life and significantly greater efficacy compared to toxin alone, antibody alone, unconjugated toxin plus antibody or even other types of toxin-antibody constructs. In a most preferred embodiment the construct comprises an anti-Trop-2 antibody conjugated to Rap, although other combinations of antibodies, antibody fragments and toxins may be used to form the subject immunotoxins. The immunotoxins are of use to treat a variety of diseases, such as cancer, autoimmune disease or immune dysfunction.

Description

The compositions and the method for using that show the immunotoxin that comprises ranpirnase (RAP) of effective cytotoxic activity
The related application of cross reference
The application requires: the 12/754th, No. 740 U.S. Patent application that on April 6th, 2010 submitted to; The 12/754th, No. 140 U.S. Patent application that on April 5th, 2010 submitted to; The 12/752nd, No. 649 U.S. Patent application that on April 1st, 2010 submitted to; The 12/731st, No. 781 U.S. Patent application that on March 25th, 2010 submitted to; The 12/644th, No. 146 U.S. Patent application of December in 2009 submission on the 22nd; With the 61/238th, No. 473 U.S. Provisional Patent Application of submitting on August 31st, 2009; The 61/266th, No. 305 U.S. Provisional Patent Application of December in 2009 submission on the 3rd; The priority of the 61/323rd, No. 960 U.S. Provisional Patent Application that the 61/316th, No. 996 U.S. Provisional Patent Application that on March 24th, 2010 submitted to and on April 14th, 2010 submit to.Each priority application integral body is by reference incorporated this paper into.
Government-funded
This working portion is supported by the fund 2R44CA108083-02A2 from the American National ICR of NIH.Federal government can have some right of the present invention.
The field
The present invention relates to preferably comprise the compositions and the method for using of toxin-antibody construct (immunotoxin) of ranpirnase (Rap); Though it will be understood by those skilled in the art that many toxin and other cytotoxic agent is compositions and the method that known and any this toxoid or cytotoxic agent can be used for the requirement protection in this area.In other embodiment preferred, said construct comprises Anti-tumor antibody, for example anti--EGP-1 (anti--Trop-2), anti--CD74, anti--CD22 or anti-CD 20.Yet; Said compositions is not limited to this and said antibody or antibody fragment with method can combine the relevant antigen of any target tissue (for example cancerous cell, B cell, T cell, autoimmune disease cell, pathogen or any other disease association target cell), is known to said antigenic antibody in this area.
In a more preferred embodiment, immunotoxin be stop-with-(DNL) construct that locks, preferably comprise the ranpirnase that is connected to antibody or antibody fragment of 4 copies.Even more preferably, toxin or other cytotoxic agent are fusion rotein, and each self-contained DDD (dimerization and stop domain) part and antibody or antibody fragment are the fusion rotein that comprises 2 AD (anchoring structure territory) part.The DDD part spontaneously forms the dimer that combines the AD part, thereby produces the cytotoxic DNL construct that is conjugated to antibody or antibody fragment that comprises 4 copies.The immunotoxin of gained shows the cytotoxic activity of efficient and can use to kill and wound the disease association cell for ill experimenter it.Said immunotoxin shows than independent parental generation antibody, independent cytokine, antibody and cytotoxic non-effectiveness of puting together the bigger anti-target cell of the cytotoxin that makes up or be conjugated to control antibodies.
Background
Ribonuclease, Rap (Lee, Exp Opin Biol Ther 2008 particularly; 8:813-27) with its more basic variant amphinase people such as (, Curr Pharm Biotechnol2008:9:215-25) Ardelt, be potential Anti-tumor agent (Lee and Raines, Biodrugs 2008; 22:53-8).Rap is at first from isolating 104 the amino acid whose singlestranded RNA enzymes of the oocyte of American leopard frog (Rana pipiens).Rap shows the in-vitro cell growth inhibition of kinds of tumor cells system and cytotoxic effect and anti-tumor in vivo activity.The Amphibian ribonuclease gets into cell through receptor-mediated endocytosis, and in case by internalization to cytosol, with regard to selectivity degraded tRNA, the inhibition and apoptotic the inducing that cause protein synthesis.
Rap has accomplished randomization IIIb clinical trial phase; This experiment is Rap+ doxorubicin and the independent effect of doxorubicin in suffering from the patient of unresectable malignant mesothe relatively; Intermittent analysis shows that the MST of combination is 12 months; And the MST of monotherapy is 10 months (Mutti and Gaudino, Oncol Rev 2008; 2:61-5).Can give patient's repetitive administration Rap and not have disadvantageous immunoreation, reversible nephrotoxicity is (people such as Mikulski, the J Clin Oncol 2002 of dose limitation property according to reports; 20:274-81; Int J Oncol 1993; 3:57-64).
Can Rap and other toxin or cytotoxin be stopped and be bonded to antibody or antibody fragment to be used for to the selected disease association cell targeted delivery of cancerous cell or autoimmune disease cell for example.The exemplary oncologic related antigen is EGP-1, is also referred to as Trop-2.
Trop-2 is an I type transmembrane protein and from people (people such as Fornaro, Int J Cancer1995; 62:610-8) and mouse cell (people such as Sewedy, Int J Cancer 1998; 75:324-30) clone.Except its function (people such as Ripani, Int J Cancer 1998 as the relevant calcium signal transducer (transducer) of tumor; 76:671-6); The expression that has shown people Trop-2 is that the tumor generation and the aggressivity of colon cancer cell is necessary; Said tumor takes place and aggressivity can effectively be reduced (people such as Wang, Mol Cancer Ther 2008 by the polyclonal antibody of the extracellular domain of anti-Trop-2; 7:280-5).
Trop-2 is as the interest that increases day by day of the therapeutic target of solid carcinoma (people such as Cubas, Biochim Biophys Acta 2009; 1796:309-14) be able to proof through other report, said report has write down crosses the Trop-2 that expresses at mammary gland (people such as Huang, Clin Cancer Res2005; 11:4357-64), colorectum (people such as Ohmachi, Clin Cancer Res2006; 12:3057-63; People such as Fang, Int J Colorectal Dis 2009; 24:875-84) and oral squamous cell (people such as Fong, Modern Pathol 2008; 21:186-91) the clinical meaning in the cancer.The prostate basal cell of expressing high-caliber Trop-2 is noticeable especially (people such as Goldstein, Proc Natl Acad Sci USA 2008 because of the up-to-date evidence of stem-like cell activity enrichment in external and the body; 105:20882-7).
Mouse-anti-Trop-2mAb, mRS7 be through using the rough film preparation that produces from the constitutional squamous cell carcinoma of people's lung of exenterate as immunogen, utilizes hybridoma technology to produce (people such as Stein, Cancer Res 1990; 50:1330-6).The immunoperoxidase staining of frozen tissue section shows: the antigen of being confirmed by mRS7 is present in lung, stomach, bladder, mammary gland, ovary, uterus and the prostatic tumor; Most of health adult tissues are anergy (people such as Stein, Int J Cancer 1993; 55:938-46).By the antigen of mRS7 identification afterwards through being shown as the 46-48kDa glycoprotein and being called epithelium glycoprotein-1 or EGP-1 (people such as Stein, Int JCancer 1994; 8:98-102), it also is called as Trop-2 (people such as Ripani, Int JCancer 1998 at document; 76:671-6).After combining target cell, mRS7 in 2 hours by internalization fast (people such as Stein, Int J Cancer 1993; 55:938-46).
In several early stage researchs, shown radiolabeled mRS7 xenograft in the nude mouse of targeting and treatment (people such as Stein, Antibody Immunoconj Radiopharm 1991 effectively; 4:703-12; People such as Stein, Cancer 1994; 73:816-23; People such as Shih, Cancer Res 1995; 55:5857s-63s; People such as Stein, J Nucl Med2001; 42:967-74; People such as Stein, Crit Rev Oncol Hematol 2001; 39:173-80).Yet this area exists thinks that to being connected to Rap or other cytotoxin disease treatment provides the needs of the RS7 of more effective reagent or the immunoconjugates of other disease target antibody (" immunotoxin ").
General introduction
The present invention relates to comprise the compositions and the method for using of the immunotoxin that is conjugated to disease target antibody or the segmental ranpirnase of antigen binding antibody (Rap) or other toxin.In some embodiment preferred, immunotoxin can have like the construct of example among Fig. 1 (being called 2L-Rap (Q)-hRS7 or 2L-Rap-hRS7), its comprise two copies be connected to humanization anti--Rap of the N-terminal of Trop-2 antibody (hRS7).Yet, it will be apparent to those skilled in the art that said immunotoxin is not limited to this and can uses the relevant or antigenic antibody of disease association of anti-other tumor known in the art.This type of immunotoxin shows the pharmacokinetics of effective cytotoxicity and raising, makes the toxic side effects of Rap reduce to minimum simultaneously.
In selectable embodiment, immunotoxin of the present invention can use stop-prepare and can comprise antibody or antigen binding antibody fragment and Rap or other toxin or cytotoxic conjugate with-(DNL) technology that locks.As employed below this paper, term " immunotoxin " can be meant the immunotoxin through the preparation of DNL technology, or the immunotoxin of example among Fig. 1.In preferred embodiments, the DNL construct comprises and is conjugated to the for example Rap of hRS7 of anti--Trop-2 antibody.Yet, one of skill in the art will appreciate that the DNL construct is not limited to this and DNL construct of the present invention and can comprises and be conjugated to ranpirnase or other toxin known in the art or the cytotoxic anti-antigenic antibody of any disease association or its fragment.
In concrete embodiment, immunotoxin can comprise humanization and resist-Trop-2 antibody or its fragment, for example hRS7 antibody; It comprises heavy chain CDR sequence C DR1 (NYGMN, SEQ ID NO:1), CDR2 (WINTYTGEPTYTDDFKG, SEQ ID NO:2) and the CDR3 (GGFGSSYWYFDV that connects the pure man antibody framework (FR) and constant region sequence; SEQ ID NO:3) and light chain CDR sequence C DR1 (KASQDVSIAVA, SEQ ID NO:4), CDR2 (SASYRYT, SEQ IDNO:5) and CDR3 (QQHYITPLT; SEQ ID NO:6) (referring to the people; For example, the 7th, 238; No. 785 United States Patent (USP)s, walk to from the 34th hurdle the 6th the 44th hurdle the 37th worked to quote incorporate this paper into).
In other concrete embodiment, immunotoxin can comprise humanization anti-CD 20 antibodies or its fragment, veltuzumab for example, and it comprises variable region of light chain CDR1 (RASSSVSYIH, SEQ ID NO:7); CDR2 (ATSNLAS, SEQ ID NO:8); And CDR3 (QQWTSNPPT, SEQ ID NO:9); And variable region of heavy chain CDR1 (SYNMH, SEQ ID NO:10); CDR2 (AIYPGNGDTSYNQKFKG, SEQ ID NO:11) and CDR3 (STYYGGDWYFDV or VVYYSNSYWYFDV, SEQ ID NO:12) (referring to; For example, the 7th, 435; No. 803 United States Patent (USP)s, walk to from the 38th hurdle the 15th the 46th hurdle the 52nd worked to quote incorporate this paper into).
In a more particular embodiment, immunotoxin can comprise ranpirnase (Rap) aminoacid sequence, this be known in this area (referring to; For example, NCBI Protein Data Bank accession number 1PU3_A is also referring to people such as Gorbatyuk; J Biol Chem 279:5772-80,2004).
In various embodiments, immunotoxin can comprise one or more and combine antigenic antibody or its fragment except that Trop-2 or CD20.In preferred embodiments, said antigen can be selected from ED-B, 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, neoplasm necrosis antigen, TNF-α, TRAIL receptor (R1 and R2), VEGFR, EGFR, PlGF, complement factor C3, C3a, C3b, C5a, C5 and the oncoprotein of 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, fibronectin.
Available exemplary antibodies includes but not limited to that hR1 (anti--IGF-1R, the 12/722nd, No. 645 U.S. Patent application of December in 2010 submission on the 3rd), hPAM4 (resist-mucin the 7th, 282; No. 567 United States Patent (USP)s), hA20 (anti-CD 20, the 7th, 251, No. 164 United States Patent (USP)s), hA19 (resist-CD19 the 7th; 109, No. 304 United States Patent (USP)s), hIMMU31 (anti--AFP, the 7th, 300, No. 655 United States Patent (USP)s), hLL1 (resist-CD74; The 7th, 312, No. 318 United States Patent (USP)s), hLL2 (resists-CD22 the 7th, 074; No. 403 United States Patent (USP)s), hMu-9 (anti--CSAp, the 7th, 387, No. 773 United States Patent (USP)s), hL243 (resist-HLA-DR the 7th; 612, No. 180 United States Patent (USP)s), hMN-14 (anti--CEACAM5, the 6th, 676, No. 924 United States Patent (USP)s), hMN-15 (resist-CEACAM6; The 7th, 541, No. 440 United States Patent (USP)s), hRS7 (resists-EGP-1 the 7th, 238; No. 785 United States Patent (USP)s) and hMN-3 (anti--CEACAM6, the 7th, 541, No. 440 U.S. Patent applications), patent that each is quoted or the embodiment of application part are incorporated this paper by reference into.It will be apparent to those skilled in the art that this tabulation is not restrictive and can uses any known antibody, like what discuss in more detail below.
The exemplary toxin that can be integrated into immunotoxin includes but not limited to bacteriotoxin, phytotoxin, ricin, Agglutinin, alpha toxin, saporin, ribonuclease (RNA enzyme), DNA enzyme I, staphylococcus enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, diphtherotoxin, PE, pseudomonas endotoxin, ranpirnase (Rap) and Rap (N69Q).The sequence of the toxin that each is quoted is that the clone of known (referring to for example ncbi database) and many exemplary toxin of encoding can be purchased acquisition from Invitrogen, American type culture collection and other source known in the art in this area.
Multiple embodiments can relate to immunotoxin of the present invention and be used to the purposes of treating or diagnosing the illness; Said disease includes but not limited to non--He Jiejin lymphomas, the B cell is 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, glioma, Walden Si Telun macroglobulinemia, cancer, melanoma, sarcoma, glioma and skin carcinoma.Cancer can be selected from oral cancer, gastrointestinal cancer, colon cancer, gastric cancer, lung road cancer, pulmonary carcinoma, breast carcinoma, ovarian cancer, carcinoma of prostate, uterus carcinoma, carcinoma of endometrium, cervical cancer, bladder cancer, cancer of pancreas, osteocarcinoma, hepatocarcinoma, carcinoma of gallbladder, renal carcinoma, skin carcinoma and carcinoma of testis.In addition, immunotoxin of the present invention can be used for treating for example nephritis (post-streptococcal nephritis), erythema nodosum, Gao An (family name) arteritis (Takayasu ' s arteritis), Addison disease (Addison ' s disease), rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture (Goodpasture ' s syndrome), thromboangiitis obliterans (thromboangitisubiteran), xerodermosteosis, primary biliary cirrhosis, struma lymphomatosa (Hashimoto ' s thyroiditis), thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris, Wei Genashi granuloma (Wegener ' s granulomatisis), membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis or the fibrosing alveolitis after acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea (Sydenham ' s chorea), myasthenia gravis, systemic lupus erythematosus (sle), lupus nephritis, rheumatic fever, polyglandular syndrome, bullous pemphigoid, diabetes, purpura,Henoch-Schonlein (Henoch-Schonlein purpura), the streptococcal infection of autoimmune disease.In certain embodiments, tried antibody and can be used for treating leukemia for example 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 and suffers from for example amyloidosis or the neurodegenerative diseases experimenter of Alzheimer for example of metabolic disease.In addition, pharmaceutical composition of the present invention can be used for treating the experimenter who suffers from the immune disorder obstacle.
Compositions of the present invention also can be used for the therapeutic treatment that infects, and wherein the immunoglobulin components specificity of immunotoxin combines pathogenic microorganism.In description of the present invention, pathogenic microorganism comprises pathogenic bacterium, virus, fungus and various parasite, but and these microorganisms of antibody targeting, their relevant product or the antigen of the damage with them.The instance of microorganism includes but not limited to: streptococcus agalactiae (Streptococcus agalactiae); Legionella pneumophilia (Legionella pneumophilia); Micrococcus scarlatinae (Streptococcus pyogenes); Escherichia coli (Escherichia coli); Gonococcus (Neisseria gonorrhoeae); Neisseria meningitidis (Neisseria meningitidis); Pn (Pneumococcus); Type B hemophilus influenza (Hemophilis influenzae B); Treponema pallidum (Treponema pallidum); Lyme disease spirillum (Lyme disease spirochetes); Bacillus pyocyaneus (Pseudomonas aeruginosa); Mycobacterium leprae (Mycobacterium leprae); Bacillus abortus (Brucella abortus); Tubercule bacillus (Mycobacterium tuberculosis); Tetanus toxin (Tetanus toxin); HIV-1;-2;-3; Hepatitis A; Hepatitis B; Hepatitis C; Hepatitis D; Rabies virus; Influenza virus; Cytomegalovirus; Herpes simplex virus I-type and II type; Human serum parvo appearance virus (Human serum parvo-like virus); Human papillomavirus; Polyoma virus; Respiratory syncytial virus (Respiratory syncytial virus); Varicella zoster virus; Hepatitis B virus; Human papillomavirus (Papilloma virus); Measles virus; Adenovirus; The human T-cell leukemia virus; Epstein-Barr virus (Epstein-Barr virus); Murine leukemia virus; Mumps virus; Vesicular stomatitis virus (Vesicular stomatitis virus); Sindbis alphavirus; Lymphocytic choriomeningitis virus (Lymphocytic choriomeningitis virus); Verrucosis poison (Wart virus); Blue tongue rims (Blue tongue virus); Sendai virus; Feline leukaemia virus (Feline leukemia virus); Reo virus; Poliovirus; Simian virus 40 (Simian virus 40); Molluscum contagiosum room tumor virus; Dengue virus; Rubella virus; Protozoacide; Plasmodium falciparum (Plasmodium falciparum); Plasmodium vivax (Plasmodium vivax); Toxoplasma gondii (Toxoplasma gondii); Trypanosoma rangeli (Trypanosoma rangeli); Schizotrypanum cruzi (Trypanosoma cruzi); Trypanosoma rhodesiense (Trypanosoma rhodesiensei); Trypanosoma bocagei (Trypanosoma brucei); Schistosoma mansoni (Schistosoma mansoni); Schistosoma japonicum (Schistosoma japanicum); Babesia bovis (Babesia bovis); Eimeria tenella (Elmeria tenella); Onchocerca caecutiens (Onchocerca volvulus); Crithidia cunninghami (Leishmania tropica); Trichinella (Trichinella spiralis); Little Taylor worm (Theileria parva); Blister band cestode (Taenia hydatigena); Taenia ovis (Taenia ovis); Taeniasis bovis (Taenia saginata); Echinococcus granulosus (Echinococcus granulosus); Mesocestoides corti (Mesocestoides corti); Mycoplasma arthritidis (Mycoplasma arthritidis); Mycoplasma hyorhinis (M.hyorhinis); Mycoplasma orale (M.orale); Mycoplasma arginini (M.arginini); Lai Shi acholeplasma (Acholeplasma laidlawii); Mycoplasma salivarium (M.salivarium) and mycoplasma pneumoniae (M.pneumoniae).Monoclonal antibody in conjunction with these pathogenic microbes is known in this area.
The accompanying drawing summary
Attached drawings forms the part of this description and is included to and is used to further specify certain embodiments of the present invention.Bonded these accompanying drawings of detailed description through the specific embodiments that provides among reference and this paper one or more can understand said embodiment better.
Fig. 1. (Q)-MOLECULE DESIGN and the size of hRS7.The schematic structure of 2L-Rap-X, wherein X is that IgG and Rap can be Rap (Q).
Fig. 2. use the cell binding curve of luminol substrate through PC-3 (A), Calu-3 (B) and the 22Rv1 (C) of ELISA acquisition.Mean fluorecence unit is mapped to concentration, and the data of utilizing Prism software analysis gained are to obtain K DValue.
The representative data that Fig. 3 .IVTT measures (A) shows that (Q)-hRS7 and rRap have suitable RNA enzymatic activity; And (B) and (Q) with rRap (left side)-the active initial rate of hRS7 (right side) to the concentration mapping of yeast tRNA to confirm kcat/Km.
Fig. 4. measure the vitro cytotoxicity that (the Q)-hRS7 of proof shows ME-180 (A) and T-47D (B) through MTS, and (the Q)-hRS7 through the CFA proof is to (C) DU-145 and (D) vitro cytotoxicity of PC-3 demonstration.Utilize Prism software analysis (A) and (B) in data to obtain the value of EC50.
(the Q)-hRS7 that proves in Fig. 5 .Calu-3 people xenograft models suppresses tumor growth (A) and increases the therapeutic efficiency of MST (B).With 1 * 10 7C alu-3 cell subcutaneous vaccination nude mouse.When tumor reaches about 0.15cm 3The time, the injection treatment mice of the 25 μ g that used in 7 days with single intravenous dosages or two minor ticks of 50 μ g.Control animals received saline.
Fig. 6 shows the RNA enzymatic activity of measuring through in vitro transcription translation algoscopy.
Fig. 7 shows competitive binding assay, and this is measured proves hLL1 and rap-hLL1 fusion rotein to WP, and anti--idiotype antibody of hLL1 has identical affinity.
Fig. 8 shows the vitro cytotoxicity of fusion rotein in the Daudi cell: the cytotoxicity of (A) measuring through the MTS algoscopy; (B) cytotoxicity of measuring through the BRdU algoscopy.
Fig. 9 shows the vitro cytotoxicity of fusion rotein in the MC/CAR cell of measuring through the MTS algoscopy.
Figure 10 shows the blood removing of 2L-Rap-hLL1-γ 4P in the first SCID mice of accepting to test.Use 88Y-DTPA-hLL1 (O) and 111In-DTPA-2L-Rap-hLL1-γ 4P () intravenous is injected the SCID mice of first acceptance experiment altogether.On the time of selecting after the administration, make mice hemorrhage through cardiac puncture, the radioactivity of counting blood sample.Average ± the S.D. (n=3) of the dosage of injecting in the data representation blood.
Figure 11 shows the lymphadenomatous treatment of Daudi that utilizes 2L-Rap-hLL1-γ 4P or constitutive protein confrontation aggressive minimum.With 1.5 * 10 7Individual Daudi cell intravenous inoculation SCID mice (8-10 mice/group).After 1 day, with the bolus injection treatment mice of specified dosage through 2L-Rap-hLL1-γ 4P.Matched group is with being equivalent to the constitutive protein matter of 50 μ g immunotoxins or only injecting with PBS.
Figure 12 shows the RNA enzymatic activity of measuring through in vitro transcription/translation algoscopy.The concentration of rRap (■), 2L-Rap-hLL1-γ 4P (▲) and hLL1-γ 4P (◆) is mapped to relative flat light emission (RLU).
Figure 13 shows that to utilize immunotoxin processed continuously or handled the vitro cytotoxicity of back through the DNL-Rap immunotoxin construct of carrying out washing treatment at 1 hour.
Figure 14 shows the vitro cytotoxicity of DNL-Rap immunotoxin construct in all cells system.
Detail
Definition
Only if point out clearly in addition, otherwise " one " or " one " can refer to " one or more ".
Term as used herein " with " with " or " can be used for conjunction (conjunctive) or extract (disjunctive).That is to say, except as otherwise noted, otherwise these two terms be to be understood that for be equal to " and/or ", except as otherwise noted.
" therapeutic agent " is atom, molecule or the chemical compound that can be used for treating disease.The instance of therapeutic agent comprises antibody, antibody fragment, peptide, medicine, toxin, enzyme, nuclease, hormone, immunomodulator, antisense oligonucleotide, siRNA (siRNA), chelating agen, boron compound, photosensitizer, dyestuff and radiosiotope.
" diagnostic agent " is atom, molecule or the chemical compound that can be used for diagnosing the illness.Useful diagnostic agent includes but not limited to radiosiotope, dyestuff (for example having biotin-Succ-PEG-DSPE complex), contrast agent, fluorescent chemicals or molecule and the reinforcing agent (for example paramagnetic ion) that is used for nuclear magnetic resonance (MRI).
This paper employed " antibody " is meant that total length (promptly; Naturally occurring or form through standard immunoassay globulin gene fragment recombination method) immunocompetence of immunoglobulin molecules (for example IgG antibody) or immunoglobulin molecules is (promptly; Specificity combines) part, like 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 antibody or its Fab that is not connected with therapeutic agent or diagnostic agent.The Fc of complete naked antibody part can provide effector function, for example complement combines and ADCC (referring to, for example, Markrides, Pharmacol Rev 50:59-87,1998).Naked antibody nationality can comprise apoptosis with other mechanism of inducing cell death.(Vaswani and Hamilton, Ann Allergy Asthma Immunol 81:105-119,1998.).
" antibody fragment " is the part of complete antibody, for example F (ab ') 2, F (ab) 2, Fab ', Fab, Fv, sFv, scFv, dAb etc.Regardless of its structure, antibody fragment the antigen discerned of bonded antigen and complete antibody identical.Antibody fragment comprises isolating fragment (" Fv " fragment of for example being made up of heavy chain and variable region of light chain) or the recombinant single chain peptide molecule of being made up of the variable region (wherein light chain is connected (" scFv albumen ") with variable region of heavy chain by the peptide connector)." single-chain antibody " (being abbreviated as " scFv " usually) is by comprising interaction to form the V of antigen-binding site HAnd V LThe polypeptide chain of domain is formed.V HAnd V LDomain is connected by the peptide of 1 to 25 amino acid residue usually.Antibody fragment also comprises double-stranded antibody, three chain antibodies and single domain antibody (dAb).
If when the dosage of being used has physiological meaning, just can be described as with " treatment effective dose " administration of antibodies or immunotoxin preparation or compositions as herein described.If the existence of reagent causes the detectable variation of the physiological status of recipient subjects, then this reagent just has physiological meaning.In concrete embodiment, if when the existence of antibody preparation causes antitumor reaction or alleviates the S&S of autoimmune disease state, this antibody preparation just has physiological meaning.The effect of physiological significance also can be to bring out the body fluid and/or the cell immune response of recipient subjects, causes growth inhibited or the death of target cell.
Antibody and antibody fragment
The technology that is used to prepare the monoclonal antibody of in fact anti-any target antigen is being known in the art.Referring to, for example, Kohler and Milstein, people's (volume) such as Nature 256:495 (1975) and Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, the 1st volume, 2.5.1-2.6.7 page or leaf (John Wiley & Sons 1991).In brief; Can be through the following monoclonal antibody that obtains: inject mice with comprising antigenic compositions; Take out spleen to obtain bone-marrow-derived lymphocyte, said bone-marrow-derived lymphocyte and myeloma cell are merged to produce hybridoma, clone hybridization tumor; Select to produce clone, cultivate generation to the clone of said antigenic antibody and from hybridoma culture separation antibody to said antigenic antibody.
Multiple perfect technology capable of using is separated and purification MAb from the hybridoma culture.This type of isolation technics comprises affinity chromatograph, size exclusion chromatography and the ion-exchange chromatography that utilizes the protein A agarose.Referring to, for example, the Coligan of 2.7.1-2.7.12 page or leaf and 2.9.1-2.9.3 page or leaf.In addition, referring to people such as Baines, " Purification of Immunoglobulin G (IgG), " in METHODS IN MOLECULAR BIOLOGY, the 10th volume, the 79-104 page or leaf (The Humana Press, Inc.1992).
Produce to behind the immunogenic antibody initial, can measure the sequence of antibody, prepare through recombinant technique subsequently.The humanization of murine antibody and antibody fragment is known with characterizing to those skilled in the art.The potential problems relevant with the immunogenicity of Mus constant region have been got rid of in use derived from the antibody component of humanized antibody, chimeric antibody or people's antibody.
Chimeric antibody
Chimeric antibody is a recombinant protein, and wherein the variable region of people's antibody is for example comprised that the variable region of mouse antibodies of the complementarity-determining region (CDR) of mouse antibodies replaces.When using to the experimenter, chimeric antibody demonstrates immunogenicity to be reduced and the stability increase.The general technology that is used to clone the rat immune globulin variable domains is disclosed in for example people such as Orlandi, among the Proc.Nat ' l Acad.Sci.USA 86:3833 (1989).The technology that is used to make up embedding antibody is known to those skilled in the art.As an example, people such as Leung, Hybridoma 13:469 (1994) is through the V of the Mus LL2 that will encode (anti--the CD22 monoclonal antibody) κAnd V HThe DNA sequence of domain with divide others κ and IgG 1The constant region domain makes up and produces the LL2 chimeric antibody.
Humanized antibody
Be used to produce the technology of humanization MAb is known in the art (referring to, for example, people such as Jones, Nature 321:522 (1986); People such as Riechmann, Nature 332:323 (1988), people such as Verhoeyen; Science 239:1534 (1988), people such as Carter, Proc.Nat ' l Acad.Sci.USA 89:4285 (1992); Sandhu, people such as Crit.Rev.Biotech.12:437 (1992) and Singer, J.Immun.150:2844 (1993)).Can be through mice CDR be come the chimeric or mouse monoclonal antibody of humanization from the corresponding variable domains that the variable heavy chain and the variable light chain of mouse immuning ball protein is transferred to people's antibody.Mice framework region (FR) in the chimeric mAb FR sequence of also choosing replaces.Since simply with mice CDR be transferred to the minimizing that causes affinity of antibody among the people FR usually or even forfeiture, therefore possibly need extra modification for the original affinity that recovers murine antibody.This can be through realizing the one or more people's residues in the FR district with acquisition with their Mus homologue is alternative to the antibody that its epi-position has the good combination affinity.Referring to, people such as Tempest for example, people such as Biotechnology9:266 (1991) and Verhoeyen, Science 239:1534 (1988).Usually, different with their Mus homologue and tightly be positioned at or those people FR amino acid residues of contacting one or more cdr amino acid residues can be alternate candidates.
People's antibody
With combined method or through the method that the transgenic animal that human immunoglobulin gene's seat transforms produce whole person's antibody is (for example, people such as Mancini, 2004, New Microbiol.27:315-28 known in the art; Conrad and Scheller, 2005, Comb.Chem.High Throughput Screen.8:117-26; Brekke and Loset, 2003, Curr.Opin.Phamacol.3:544-50).Gene also capable of using or chromosome infection protocol and phage display technology (all said technology all are known in this area) make up whole person's antibody.Referring to people such as for example McCafferty, Nature 348:552-553 (1990).The expection of such whole person's antibody show than chimeric antibody or humanized antibody still less side effect and as endogenous people's antibody basically, work in vivo.In certain embodiments, require the method for protection and process can use the people's antibody that produces through this type of technology.
In an alternate embodiment, phage display technology can be used for producing people's antibody (for example, Dantas-Barbosa etc., 2005, Genet.Mol.Res.4:126-40).People's antibody can or show particular disease states such as the philtrum of cancer preparation (Dantas-Barbosa etc., 2005) from the normal person.The favourable aspect that from diseased individuals, makes up people's antibody is that the full storehouse of circulating antibody maybe be to having preference property to the antigenic antibody of disease association.
In a limiting examples of the method, the phage that Dantas-Barbosa etc. (2005) have made up from human Fab's antibody fragment of osteosarcoma patient shows the library.Generally speaking, total RNA is available from CBL (Id.).Clone's reorganization Fab from μ, γ and the full storehouse of κ chain antibody, and be inserted into phage demonstration library (Id.).RNA can be converted into cDNA to heavy chain and the special primer of light chain immunoglobulin sequences through using, and be used to prepare the FabcDNA library (people such as Marks, 1991, J.Mol.Biol.222:581-97).Library construction press Andris-Widhopf etc. (2000, at Phage Display Laboratory Manual, Barbas etc. (volume); The 1st edition; Cold Spring Harbor Laboratory Press, Cold Spring Harbor is among the NY pp.9.1 to 9.22) carry out.Final Fab fragment digests with restriction endonuclease, inserts phage genome then and shows the library to prepare said phage.This type of library can use standard phage display packing known in the art screen (referring to, for example, Pasqualini and Ruoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart.J.Nucl.Med.43:159-162).
Can implement phage in a variety of forms and show, about their summary, referring to for example Johnson and Chiswell, Current Opinion in Structural Biology3:5564-571 (1993).External activatory B cell also capable of using produces people's antibody.Referring to, the 5th, 567, No. 610 and the 5th, 229, No. 275 United States Patent (USP)s, with said patent by reference integral body incorporate this paper into.It will be understood by those skilled in the art that these technology are any known methods that are used to prepare and screen people's antibody or antibody fragment exemplary and capable of using.
In another alternate embodiment, use the immunization experiment scheme of standard, can be used for producing to the antibody of any immune target basically with the transgenic animal that produce people's antibody through genetic engineering modified.The method that is used for obtaining people's antibody from transgenic animal is by people such as Green, Nature Genet.7:13 (1994), and people such as Lonberg, people such as Nature 368:856 (1994) and Taylor, Int.Immun.6:579 (1994) discloses.The non-limiting example of this system is Abgenix (Fremont; CA)
Figure BDA0000139409280000151
(for example; People such as Green; 1999, J.Immunol.Methods 231:11-23).At with similar animal in; The mouse antibodies gene is by inactivation; And replace with the functional human antibody gene, but the immune remainder of Mus still is kept perfectly.
Figure BDA0000139409280000161
transformed the YAC (yeast artificial chromosome) that plants system's configuration; This YAC comprises groups of people IgH and IgK locus; The most of zone that comprises variable region sequences, and auxiliary gene and regulating and controlling sequence.Storehouse, people variable region can be used for preparing the B cell that produces antibody, and the B cell can merge to hybridoma through known technology.
Figure BDA0000139409280000162
with the target antigen immunity will produce people's antibody through normal immunoreation, and said people's antibody can be gathered in the crops and/or produce through above-mentioned standard technique.Have
Figure BDA0000139409280000163
of a plurality of kinds of systems available, each plants system all can produce different classes of antibody.People's antibody that transgenic produces has demonstrated the treatment potentiality, and has kept the pharmacokinetic properties (Green etc., 1999) of normal person's antibody.Those skilled in the art will understand that; Require the compositions and the method for protection to be not limited to use
Figure BDA0000139409280000164
system, but can use any transgenic animal of the genetic engineering modified people's of the generation antibody of process.
Antibody fragment
Known technology capable of using produces the antibody fragment of identification defined epitope.Antibody fragment is the antigen-binding portion thereof F (ab ') for example of antibody 2, Fab ', F (ab) 2, Fab, Fv, sFv etc.Can produce F (ab ') through the pepsin antibody molecule of degrading 2Fragment, and can be through reduction F (ab ') 2Segmental disulphide bridges produces Fab ' fragment.Selectively, can make up Fab ' expression library (people such as Huse, 1989, Science is 246:1274-1281) to allow fast and easily to identify the having specific monoclonal Fab ' fragment of expectation.Can produce F (ab) through papain digestion antibody 2Fragment, and also obtained the Fab fragment originally through disulphide.
Strand Fv molecule (scFv) comprises VL domain and VH domain.VL and VH domain associate to form the target binding site.These two domains are further covalently bound through peptide connector (L).Be used to prepare the scFv molecule and be disclosed in the 4th, 704, No. 692 United States Patent (USP)s with the method that designs suitable peptide connector; The 4th, 946, No. 778 United States Patent (USP)s; R.Raag and M.Whitlow, " Single Chain Fvs. " FASEB the 9th volume: 73-80 (1995) and R.E.Bird and B.W.Walker, " Single Chain Antibody Variable Regions; " TIBTECH, the 9th volume: among the 132-137 (1991).
The technology that is used to produce single domain antibody (DAB) also is known in this area, and is disclosed in (incorporating this paper by reference into) like people (2006, Prot Express Purif 51:253-259) such as for example Cossins.
Can be through the Proteolytic enzyme full length antibody or through preparing antibody fragment escherichia coli (E.coli) or the said segmental DNA of another kind of Su Zuzhong expression coding.Conventional method capable of using obtains antibody fragment through pepsin or papain degraded full length antibody.These class methods are by for example Goldenberg, and the reference material that comprises among the 4th, 036, No. 945 and the 4th, 331, No. 647 United States Patent (USP)s and this paper is described.In addition, referring to people such as Nisonoff, Arch Biochem.Biophys.89:230 (1960); Porter, Biochem.J.73:119 (1959), people such as Edelman, in METHODS IN ENZYMOLOGY the 1st volume, the Coligan of the 422nd page (Academic Press 1967) and 2.8.1-2.8.10 and 2.10.-2.10.4 page or leaf.
Known antibody
The antibody that uses can be purchased acquisition from many known sources.For example, a plurality of antibody-secreting type hybridoma strains can available from American type culture collection (ATCC, Manassas, VA).The antibody of a large amount of anti-various disease targets (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 to be used for the method and composition of requirement protection.Referring to, for example, 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, the embodiment part is incorporated this paper by reference into.These only are that exemplary and a lot of other antibody of kind and their hybridoma are known in this area.It will be understood by those skilled in the art that anti-antigenic antibody sequence of almost any disease association or antibody-secreting type hybridoma can obtain through searching for ATCC, NCBI and/or USPTO data base simply with regard to the antibody of anti-selected disease association target.Can use the antigen binding structural domain of standard technique amplification clone's well known in the art antibody,, connect into expression vector then, be transferred to appropriate host cell, and be used for protein production its cutting.
Immunoconjugates
In certain embodiments, can antibody or its fragment be conjugated to one or more therapeutic agents or diagnostic agent.Therapeutic agent needn't be identical but can be different, for example medicine and radiosiotope.For example, can with 131I mixes the tyrosine medicine amino with the ε that is connected to lysine residue of antibody or fusion rotein.Also can therapeutic agent and diagnostic agent be connected to for example reduced form SH group and/or saccharide side chain.The many methods that are used to prepare the covalently or non-covalently conjugate of therapeutic agent or diagnostic agent and antibody or fusion rotein are any known like this method known and capable of using in this area.
Can be through connecting therapeutic agent or diagnostic agent on the hinge region that is formed on the reduced form antibody component of disulfide bond.Selectively, can use the isodigeranyl functional cross-link agent for example N-succinyl group 3-(2-pyridine dithio) propionic ester (SPDP) connect this type of reagent.People such as Yu, Int.J.Cancer 56:244 (1994).Being used for such general technology of puting together is being known in the art.Referring to, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); People such as Upeslacis; " Modification of Antibodies by Chemical Methods, " in MONOCLONAL ANTIBODIES:PRINCIPLES AND APPLICATIONS, people such as Birch (volume); The 187-230 page or leaf (Wiley-Liss, Inc.1995); Price; " Production and Characterization of Synthetic Peptide-Derived Antibodies; " In MONOCLONAL ANTIBODIES:PRODUCTION; ENGINEERING AND CLINICAL APPLICATION, people such as Ritter (volume), 60-84 page or leaf (Cambridge University Press 1995).Selectively, can partly put together therapeutic agent or diagnostic agent through the saccharide in the Fc district of antibody.Glycosyl can be used for increasing the loading with the bonded identical reagent of sulfydryl, or saccharide partly can be used for combining different therapeutic agents or diagnostic agent.
Being used for peptide is known via the method that the saccharide of antibody partly is conjugated to antibody component to those skilled in the art.Referring to, for example, people such as Shih, Int.J.Cancer 41:832 (1988); People such as Shih, people such as Int.J.Cancer 46:1101 (1990) and Shih, U.S.Patent No.5,057,313, with said document by reference integral body incorporate this paper into.Conventional method comprises makes antibody component with oxidized form saccharide part and the carrier polymer reaction with at least one unhindered amina function.This reaction causes producing initial schiff bases (imines) bonding, and it can obtain to stablize to form final conjugate through being reduced into secondary amine.
If being used as the antibody of the antibody component of immunoconjugates is antibody fragment, the Fc district can not exist so.Yet, maybe saccharide partly be introduced the variable region of light chain of full length antibody or antibody fragment.Referring to, for example, people such as Leung, J.Immunol.154:5919 (1995); People such as Hansen, U.S.Patent No.5,443,953 (1995), people such as Leung, the 6th, 254, No. 868 United States Patent (USP)s, with said document and patent by reference integral body incorporate this paper into.Genetic engineering modified saccharide partly is used to connect therapeutic agent or diagnostic agent.
In certain embodiments, can chelating agen be connected to antibody, antibody fragment or fusion rotein, be used for for example radionuclide of chelating therapy agent or diagnostic agent then.Exemplary chelating agen includes but not limited to DTPA (for example Mx-DTPA), DOTA, TETA, NETA or NOTA.Be used for chelating agen put together and use its with metal or other part be connected to method of protein be known in the art (referring to, for example, the 7th, 563, No. 433 United States Patent (USP)s for example, the embodiment part is incorporated this paper by reference into).
In certain embodiments, can a plurality of chelation groups that are used for coupled ion be connected to said long-tail through radioactive metal or paramagnetic ion being connected to protein or peptide with macrurous reagent reacting.Such tail can be that for example but poly-D-lysine, polysaccharide or other have the derive polymer or the derivative chain of side group to polymer; Said side group can combine the known chelation group that can be used for this purposes, for example ethylenediaminetetraacetic acid (EDTA), diethylene triamine pentacetic acid (DTPA) (DTPA), porphyrin, polyamines, crown ether, two-thiosemicarbazones, gather oxime (polyoxime) etc.
Can chelating agen be connected directly to antibody or peptide, for example as disclosed in the 4th, 824, No. 659 United States Patent (USP)s (it incorporates this paper by reference into).Useful especially metal-chelating agen combination comprises and 60 to 4, the diagnosis isotope that is used for radiological imaging (radioimaging) in the general energy range of 000keV (for example 125I, 131I, 123I, 124I, 62Cu, 64Cu, 18F, 111In, 67Ga, 68Ga, 99mTc, 94mTc, 11C, 13N, 15O, 76Br) the 2-benzyl-DTPA and monomethyl and the cyclohexyl analog that use together.When with on-radiation metal for example when manganese, ferrum and gadolinium complexation, identical chelating agen can be used for MRI.Macrocyclic chelants like NOTA, DOTA and TETA, can be used for multiple metal and radiometal, particularly is respectively applied for gallium, yttrium and copper radionuclide.Through size, can make this metalloid-chelating agen complex highly stable according to the metal target adjustable ring.The chelating agen that can comprise other lopps type, Macrocyclic polyester for example, it is used for stably combining nucleic for example by concern 223Ra (for RAIT).
Recently, disclose and be used for the PET scanning technique 18The method of F-labelling, for example through F-18 and metal or other atom for example the reaction of aluminum carry out.Can with 18F-Al conjugate and chelation group be DOTA, NOTA or NETA complexation for example, but said chelate group is connected directly to antibody or in preparatory targeted approach, is used to the construct of labelling targeting.Such F-18 labelling technique is disclosed in the 7th, 563, in No. 433 United States Patent (USP)s, incorporates the embodiment part into this paper by reference.
The immunotoxin that comprises ranpirnase (Rap)
Rap to puting together or merging of cancer target antibody or antibody fragment be the method for promising its effectiveness of enhancing, as at first for LL2-ranpirnase (people such as Newton, Blood2001; 97:528-35) (the chemically conjugated thing that comprises Rap and mouse-anti-CD22 monoclonal antibody (mAb)) and subsequently for 2L-Rap-hLL1-γ 4P (referring to; For example; Embodiment 2 hereinafter) (comprise Rap and humanization anti--fusion rotein of CD74mAb (people such as Stein, Blood2004; 104:3705-11)) proved.
The method that is used to produce 2L-Rap-hLL1-γ 4P allows us to develop the immunotoxin (being commonly referred to 2L-Rap-X) of a series of structural similarities; It is all by two Rap molecular compositions, and each molecule is connected to the N end of a L chain in the purpose antibody (X) through flexible connection body.We also (are called 2LRap (Q)-X) through substitute another serial immunotoxin that Rap produces same design with the non-glycosylated form of its Rap (be called Rap (Q), be changed into Gln (or Q, single-letter code) with the potential glycosylation site on the expression Asn69).For these two series, we are prepared as IgG1 (γ 1) or IgG4 (γ 4) with IgG, and make its formation that stops IgG4 half point (Aalberse and Schuurman, Immunology 2002; 105:9-19), we convert the serine residue in the hinge region (S228) of IgG4 to proline (γ 4P).The exemplary results of 2L-Rap-X or 52L-Rap (Q)-X is shown among Fig. 1.This design is depended on the needs of the pyroglutamic acid residue on the N end of Rap (it is essential that this residue will have complete function for the RNA enzyme) (people such as Liao, Nucleic Acids Res 2003; 31:5247-55).
In order to develop the function of mRS7, migrate on human IgG1's framework (people such as Qu, Methods2005 through complementary determining region with mRS7 as the potential therapeutic agent of the solid carcinoma of expressing Trop-2; 36:84-95) with merge to Rap (Q), thereby cause being abbreviated as hereinafter 2L-Rap (Q)-hRS7 of (Q)-hRS7 to prepare humanization RS7 (hRS7).
In the work of describing among the embodiment hereinafter; We can produce in mammalian hosts through demonstration (Q)-hRS7 (i) and can be purified to homogenizing; The RNA enzymatic activity that (ii) keeps binding specificity and affinity and the Rap of hRS7, the growth that (iii) on nanomolar concentration, suppresses human lung cancer's xenograft in the propagation of the cancerous cell line of the multiple expression of external inhibition Trop-2 and the (iv) body shows: Rap or Rap (Q) to the N-terminal fusion of cancer target mAb be the method that is proved to be the novel immunotoxin of effective and polyenergic generation.
Those skilled in the art will admit, be suitable for cytotoxicity RNA enzyme of the present invention and partly comprise the polypeptide and its all enzymatic activity variants with natural ranpirnase structure.This quasi-molecule advantageously has for the seemingly essential N-terminal pyroglutamic acid residue of RNA enzymatic activity and is not suppressed by the mammalian rna enzyme inhibitor basically.The nucleic acid of coding natural cytotoxicity RNA enzyme can be through the clone and the restriction of suitable sequence, or use utilizes the DNA cloning of polymerase chain reaction (PCR) to prepare.The aminoacid sequence of American leopard frog ranpirnase can be available from people such as Ardelt; J.Biol.Chem.; 256:245 (1991), and the cDNA sequence of the conservative variation of modifying of natural ranpirnase or its of encoding can through be used for the humanized en bloc of hLL2 V-gene assembling method similar methods and come gene synthetic.(people such as Leung, Mol.Immunol., 32:1413,1995).The method for preparing cytotoxicity RNA enzyme variants this area be known and ability those skilled in the art within.
Selectively, the nucleic acid of Codocyte toxicity RNA enzyme or its variant can be external synthetic.Chemical synthesis produces single stranded oligonucleotide.Can through with complementary sequence hybridization, or through use short primer and with said strand as template, utilize the polymerization of archaeal dna polymerase that this single stranded oligonucleotide is changed into double-stranded DNA.Though chemosynthesis is suitable for the sequence of about 100 bases most, can obtain longer sequence through connecting shorter sequence.Embodiment 2 hereinafter provides an illustrative methods that is used to obtain cytotoxicity RNA enzyme gene.
Stop and (DNL) method that locks
" stop-with-lock " (DNL) method utilized particular proteins interphase interaction (people such as Baillie, the FEBS Letters.2005 of generation between the anchoring structure territory (AD) of adjusting (R) subunit and A-kinases anchorin (AKAP) of the protein kinase (PKA) that cAMP relies on; 579:3264.Wong and Scott, Nat.Rev.Mol.Cell Biol.2004; 5:959).PKA (in by one of signal transduction path that passes through second message,second messenger cAMP and the combination triggering of R subunit of scrutiny, playing central role) separates (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 are kept inactive form (Taylor, J.Biol.Chem.1989 through the R subunit; 264:8443).The isozyme class of finding PKA has two types of R subunits (RI and RII), and each class has α and β isotype (Scott, Pharmacol.Ther.1991; 50:123).The R subunit is only separated with stable dimer, and has shown that the dimerization domain forms (people such as Newlon, Nat.Struct.Biol.1999 by preceding 44 aminoterminal residues; 6:222).Cause the combining of cAMP and R subunit being used for the release of the active catalytic subunit of wide spectrum activity of serine/threonine kinases; Said release is directed to selected substrate (people such as Scott, J.Biol.Chem.1990 through PKA via the compartmentation of the stop of itself and AKAP; 265; 21561).
Because an AKAP, microtubule bindin 2 was able to characterize (people such as Lohmann, Proc.Natl.Acad.Sci USA.1984 in 1984; 81:6723); In yeast to human species scope, identified and had the different subcellular locations of being positioned to of the different structure AKAP of (comprising plasma membrane, actin cytoskeleton, nucleus, mitochondrion and endoplasmic reticulum) (Wong and Scott, Nat.Rev.Mol.Cell Biol.2004 above 50 kinds; 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 reported 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 combine dimerization R subunit.For people RII, hydrophobic surface (Colledge and Scott, Trends Cell Biol.1999 that the AD combination is formed by 23 n terminal residues; 6:216).Therefore, the dimerization domain of people RII α all is arranged in 44 aminoacid sequences of identical N-terminal (people such as Newlon, Nat.Struct.Biol.1999 with the AKAP binding structural domain; 6:222; People such as Newlon, EMBO are J.2001; 20:1651), said aminoacid sequence is called DDD in this article.
As the DDD of the people RII α of link block (Linker Modules) and the AD of AKAP
We have developed such platform technology; Its DDD and proteic AD of AKAP that utilizes people PKA RII α stops any combination of the entity that is called A and B hereinafter to non-covalent complex being used for as a pair of good link block, and non-covalent complex can be through further locking in the structure of stable constraint with the formation that promotes disulfide bond on the strategic location of cysteine residues being introduced DDD and AD.The conventional method of " stop-with-lock " method is following.Through the DDD sequence is connected the entity A that (result is created in first component that hereinafter is called a) makes up with the precursor of A.Because the DDD sequence can cause dimeric spontaneous formation, so A can be by a 2Form.Can make up entity B through the AD sequence is connected (result is created in second component that hereinafter is called b) with the precursor of B.a 2In the dimerization primitive of the DDD that comprises can produce and be used for the bonded stop of the AD sequence site that comprises with b, thereby promote a 2With the easy association of b to form by a 2The trimerization complex that b forms.Such binding events irreversibly carries out because of subsequent reactions; Said subsequent reactions has been protected said two entities through the disulphide bridges covalency; Said reaction takes place based on the principle of effective localized concentrations very effectively; Because initial binding interactions should make the reactive mercapto that places on DDD and the AD near (people such as Chmura, Proc.Natl.Acad.Sci.USA.2001; 98:8480) connect so that carry out locus specificity.In certain embodiments, a 2Subunit can comprise 2 identical effector parts, and for example antibody, antibody fragment or cytotoxin are connected to identical DDD sequence separately.Trimerization a 2The b complex can comprise first effector part of two copies and the second effector part of a copy.
Through connecting DDD and the AD away from the functional group of precursor, this type of locus specificity connects the original activity that expection keeps precursor.This method is module in essence and can be used for locus specificity potentially and covalently be connected many kinds of materials.The DNL method is disclosed in the 7th, 550, No. 143; The 7th, 521, No. 056; The 76th, 534, No. 866; In the 7th, 527, No. 787 and the 7th, 666, No. 400 United States Patent (USP)s, the embodiment part of each patent is incorporated this paper by reference into.Though the DNL complex can comprise the trimer structure in preferred embodiments, in selectable embodiment, other stoichiometry also is possible, for example the antibody or the antibody fragment of the toxin moiety of 4 copies and 1 copy.
In preferred embodiments, effector partly is partly to be connected and the protein or the peptide that form fusion rotein or peptide, more preferably antibody, antibody fragment or toxin with DDD or AD.The several different methods that is used to prepare fusion rotein is known, comprises that nucleic acid is synthetic, hybridization and/or amplification to be to produce the synthetic double-strandednucleic acid of coding purpose fusion rotein.Standard molecular biological technique capable of using (referring to, people such as Sambrook for example, Molecular Cloning, A laboratorymanual, the 2nd edition, 1989) this type of double-strandednucleic acid inserted be used for the expression vector that fusion rotein produces.In this type of embodiment preferred, can AD and/or DDD part be connected with the N-terminal or the C-terminal of effect protein or peptide.Yet the site that those skilled in the art will know that AD or DDD part to the connection of effector part can be depending on the chemical property of effector part and changes, and the part of effector part involves its physiologically active.In most preferred embodiment, the connection of AD or DDD part to antibody or antibody fragment can take place on the C-terminal of heavy chain subunit, end relatively from the molecule of antigen-binding site.Yet, like what hereinafter discussed, also can use N-terminal connection to antibody or antibody fragment, keep antigen-binding activity simultaneously.The locus specificity that can use technology known in the art for example to use bivalence cross-linking reagent and/or other chemically conjugated technology to carry out multiple effector part connects.
DDD and AD sequence variants
In certain embodiments, AD and the DDD sequence that is integrated into immunotoxin DNL construct comprises DDD1 and the aminoacid sequence of AD1 hereinafter.In a more preferred embodiment, AD and DDD sequence comprise the aminoacid sequence of DDD2 and AD2, and said sequence promotes the formation of disulfide bond between DDD and the AD part through design.
DDD1
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：13)
DDD2
CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：14)
AD1
QIEYLAKQIVDNAIQQA(SEQ?ID?NO：15)
AD2
CGQIEYLAKQIVDNAIQQAGC(SEQ?ID?NO：16)
It will be understood by those skilled in the art that DDD1 and DDD2 comprise the DDD sequence of the people RII alpha form of PKA.Yet in selectable embodiment, DDD and AD part can be based on the DDD sequence and corresponding AKAP sequence of the people RI alpha form of PKA, and is illustrational like institute among hereinafter DDD3, DDD3C and the AD3.
DDD3
SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK(SEQ?ID?NO：17)
DDD3C
MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK(SEQ?ID?NO：18)
AD3
CGFEELAWKIAKMIWSDVFQQGC(SEQ?ID?NO：19)
Also can design and utilize based on the selectable DDD part of other of known people RI β and RII beta amino acids sequence (referring to, for example, NCBI accession number NP_001158233 and NP_002727, sequence hereinafter).
People PKA RI beta amino acids sequence
MASPPACPSE?EDESLKGCEL?YVQLHGIQQV?LKDCIVHLCI?SKPERPMKFL
REHFEKLEKE?ENRQILARQK?SNSQSDSHDE?EVSPTPPNPV?VKARRRRGGV
SAEVYTEEDA?VSYVRKVIPK?DYKTMTALAK?AISKNVLFAH?LDDNERSDIF
DAMFPVTHIA?GETVIQQGNE?GDNFYVVDQG?EVDVYVNGEW?VTNISEGGSF
GELALIYGTP?RAATVKAKTD?LKLWGIDRDS?YRRILMGSTL?RKRKMYEEFL
SKVSILESLE?KWERLTVADA?LEPVQFEDGE?KIVVQGEPGD?DFYIITEGTA
SVLQRRSPNE?EYVEVGRLGP?SDYFGEIALL?LNRPRAATVV?ARGPLKCVKL
DRPRFERVLG?PCSEILKRNI?QRYNSFISLT?V(SEQ?ID?NO：20)
People PKA RI beta amino acids sequence
MSIEIPAGLT?ELLQGFTVEV?LRHQPADLLE?FALQHFTRLQ?QENERKGTAR
FGHEGRTWGD?LGAAAGGGTP?SKGVNFAEEP?MQSDSEDGEE?EEAAPADAGA
FNAPVINRFT?RRASVCAEAY?NPDEEEDDAE?SRIIHPKTDD?QRNRLQEACK
DILLFKNLDP?EQMSQVLDAM?FEKLVKDGEH?VIDQGDDGDN?FYVIDRGTFD
IYVKCDGVGR?CVGNYDNRGS?FGELALMYNT?PRAATITATS?PGALWGLDRV
TFRRIIVKNN?AKKRKMYESF?IESLPFLKSL?EFSERLKVVD?VIGTKVYNDG
EQIIAQGDSA?DSFFIVESGE?VKITMKRKGK?SEVEENGAVE?IARCSRGQYF
GELALVTNKP?RAASAHAIGT?VKCLAMDVQA?FERLLGPCME?IMKRNIATYE
EQLVALFGTN?MDIVEPTA(SEQ?ID?NO：21)
In other selectable embodiment, the different sequence variants of AD and/or DDD part can be used for the structure of immunotoxin DNL construct.Structure-the functional relationship of AD and DDD domain is Investigational theme always.(referring to, for example, people such as Burns-Hamuro, 2005, Protein Sci 14:2982-92; People such as Carr, 2001, J Biol Chem276:17332-38; People such as Alto, 2003, Proc Natl Acad Sci USA 100:4445-50; People such as Hundsrucker, 2006, Biochem J 396:297-306; People such as Stokka, 2006, Biochem J 400:493-99; People such as Gold, 2006, Mol Cell 24:383-95; People such as Kinderman, 2006, Mol Cell 24:397-408, the full copy of each document is incorporated this paper by reference into).
For example; People such as Kinderman (2006) have checked the crystal structure of AD-DDD binding interactions and have drawn people DDD sequence and comprised many conservative amino acid residues; Said amino acid residue is particular importance in dimer formation and AKAP combination, in following sequence, underlines.(referring to people such as Kinderman, Fig. 1 of 2006, said document is incorporated this paper by reference into).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, however can be alternative to combine not too critical residue to carry out conserved amino acid for dimerization and AKAP.Therefore, the potential selectable DDD sequence that is used for making up the DNL construct is shown in SEQ ID NO:22, and wherein " X " expression conserved amino acid substitutes.Conserved amino acid sequence substitutes and discusses in more detail hereinafter, but can comprise that for example asparagicacid residue substitutes glutaminic acid residue or leucine or the alternative isoleucine residue of valine residue etc.This type of conserved amino acid substitutes and is being known in the art.
People DDD sequence from PKA
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：13)
XXIXIXXXLXXLLXXYXVXVLXXXXXXLVXFXVXYFXXLXXXXX(SEQ?ID?NO：22)
People such as Alto (2003) have carried out the bioinformatic analysis of the proteic AD sequence of multiple AKAP to design the RII selectivity AD sequence that is called AKAP-IS that hereinafter shows, it has the binding constant to DDD of 0.4nM.The AKAP-IS sequence is designed to combine the peptide antagonists of the AKAP of PKA.The residue that wherein substitutes in the bonded AKAP-IS sequence tend to reduce with DDD underlines in following sequence.Therefore, the variant that it will be understood by those skilled in the art that the function that can bring into play the DNL construct is by SEQ ID NO:23 indication, and wherein " X " is that conserved amino acid substitutes.
The AKAP-IS sequence
QIEYLAKQIVDNAIQQA(SEQ?ID?NO：15)
XXXXXAXXIVXXAIXXX(SEQ?ID?NO：23)
Similarly, Gold (2006) utilizes crystallography and peptide screening to develop the SuperAKAP-IS sequence that hereinafter shows, it shows that the selectivity to the RII isotype of PKA exceeds selectivity 5 one magnitude to the RI isotype.Underlined residue is represented the position with respect to the amino acid replacement of AKAP-IS sequence, the combining of the DDD part of said alternative increase and RII α.In this sequence, it is residue numbers 20 that N-terminal Q residue is numbered as residue 4 and C-terminal A residue.Wherein can substitute with influence is residue 8,11,15,16,18,19 and 20 people such as (, 2006) Gold to the residue of the affinity of RII α.Be expected in some selectable embodiment, available SuperAKAP-IS sequence replacing AKAP-IS AD partial sequence is with preparation cytotoxicity DNL construct.Other selectable sequence that can be used to alternative AKAP-IS AD sequence is shown in hereinafter.Underline sign substituting with respect to the AKAP-IS sequence.Expection, about the AKAP-IS sequence, AD partly also can comprise extra N-terminal residue cysteine and glycine and C-terminal residue glycine and cysteine.
SuperAKAP-IS
QIEYVAKQIVDYAIHQA(SEQ?ID?NO：24)
Selectable AKAP sequence
QIEYKAKQIVDHAIHQA(SEQ?ID?NO：25)
QIEYHAKQIVDHAIHQA(SEQ?ID?NO：26)
QIEYVAKQIVDHAIHQA(SEQ?ID?NO：27)
Fig. 2 of people such as Gold disclose hereinafter show from the proteic extra DDD-binding sequence of multiple AKAP.
RII-specificity AKAP
AKAP-KL
PLEYQAGLLVQNAIQQAI(SEQ?ID?NO：28)
AKAP79
LLIETASSLVKNAIQLSI(SEQ?ID?NO：29)
AKAP-Lbc
LIEEAASRIVDAVIEQVK(SEQ?ID?NO：30)
RI-specificity AKAP
AKAPce
ALYQFADRFSELVISEAL(SEQ?ID?NO：31)
RIAD
LEQVANQLADQIIKEAT(SEQ?ID?NO：32)
PV38
FEELAWKIAKMIWSDVF(SEQ?ID?NO：33)
Dual-specificity AKAP
AKAP7
ELVRLSKRLVENAVLKAV(SEQ?ID?NO：34)
MAP2D
TAEEVSARIVQVVTAEAV(SEQ?ID?NO：35)
DAKAP1
QIKQAAFQLISQVILEAT(SEQ?ID?NO：36)
DAKAP2
LAWKIAKMIVSDVMQQ(SEQ?ID?NO：37)
People such as Stokka (2006) have also developed the peptide competitor of the AKAP of the combination PKA that hereinafter shows.Said peptide antagonists is named as Ht31, RIAD and PV-38.The Ht-31 peptide shows the affinity of bigger RII isotype to PKA, and RIAD and the bigger affinity to RI of V-38 demonstration.
Ht31
DLIEEAASRIVDAVIEQVKAAGAY(SEQ?ID?NO：38)
RIAD
LEQYANQLADQIIKEATE(SEQ?ID?NO：39)
PV-38
FEELAWKIAKMIWSDVFQQC(SEQ?ID?NO：40)
People such as Hundsrucker (2006) have also developed other peptide competitor of the AKAP that combines PKA, have the binding constant to the DDD of the RII form of PKA that is low to moderate 0.4nM.In the people's such as Hundsrucker that the sequence of multiple AKAP antagonistic peptides is provided in hereinafter to duplicate the table 1.
Table 1AKAP peptide sequence
AKAPIS representes synthetic RII subunit binding peptide.All other peptides derived from shown in the RII binding structural domain of AKAP.
Peptide sequence
AKAPIS QIEYLAKQIVDNAIQQA(SEQ?ID?NO：15)
AKAPIS-P QIEYLAKQIPDNAIQQA(SEQ?ID?NO：41)
Ht31 KGADLIEEAASRIVDAVIEQVKAAG(SEQ?ID?NO：42)
Ht31-P KGADLIEEAASRIPDAPIEQVKAAG(SEQ?ID?NO：43)
AKAP7δ-wt-pep PEDAELVRLSKRLVENAVLKAVQQY(SEQ?ID?NO：44)
AKAP7δ-L304T-pep?PEDAELVRTSKRLVENAVLKAVQQY(SEQ?ID?NO：45)
AKAP7δ-L308D-pep?PEDAELVRLSKRDVENAVLKAVQQY(SEQ?ID?NO：46)
AKAP7δ-P-pep PEDAELVRLSKRLPENAVLKAVQQY(SEQ?ID?NO：47)
AKAP7δ-PP-pep PEDAELVRLSKRLPENAPLKAVQQY(SEQ?ID?NO：48)
AKAP7δ-L314E-pep?PEDAELVRLSKRLVENAVEKAVQQY(SEQ?ID?NO：49)
AKAP1-pep EEGLDRNEEIKRAAFQIISQVISEA(SEQ?ID?NO：50)
AKAP2-pep LVDDPLEYQAGLLVQNAIQQAIAEQ(SEQ?ID?NO：51)
AKAP5-pep QYETLLIETASSLVKNAIQLSIEQL(SEQ?ID?NO：52)
AKAP9-pep LEKQYQEQLEEEVAKVIVSMSIAFA(SEQ?ID?NO：53)
AKAP10-pep NTDEAQEELAWKIAKMIVSDIMQQA(SEQ?ID?NO：54)
AKAP11-pep VNLDKKAVLAEKIVAEAIEKAEREL(SEQ?ID?NO：55)
AKAP12-pep NGILELETKSSKLVQNIIQTAVDQF(SEQ?ID?NO：56)
AKAP14-pep TQDKNYEDETQVALALVEDVINYA(SEQ?ID?NO：57)
Rab32-pep ETSAKDNINIEEAARFLVEKILVNH(SEQ?ID?NO：58)
The residue conservative at the proteic AD domain of different AKAP camber underlines sign hereinafter through the following AKAP IS sequence of reference.Except the interpolation of C-terminal alanine residue, said residue is with observed identical by people such as Alto (2003).(, incorporating said document into this paper by reference) referring to Fig. 4 of people such as Hundsrucker (2006).Sequence with peptide antagonists of extra high affinity to RII DDD sequence is the sequence of AKAP-IS, AKAP7 δ-wt-pep, AKAP7 δ-L304T-pep and AKAP7 δ-L308D-pep.
AKAP-IS
QIEYLAKQIVDNAIQQA(SEQ?ID?NO：15)
People such as Carr (2001) have checked the different AKAP from people and non-human protein's matter to combine the sequence homology degree between the DDD sequence and identified as if topnotch is guarded in different DDD parts in the DDD sequence residue.These can underline sign hereinafter through reference man PKA RII α DDD sequence.Conservative especially residue is further indicated by italics.Said residue with propose by people such as Kinderman (2006) overlapping for combining those important residues of AKAP albumen, but not identical with it.Therefore, the potential DDD sequence of indication in SEQ ID NO:59, wherein " X " expression conserved amino acid substitutes.
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：13)
XHIXIPXGLXELLQGYTXEVLRXQPXDLVEFAXXYFXXLXEXRX(SEQ?ID?NO：59)
Those skilled in the art will understand that; Generally speaking; These are being can preferably produce in the amino acid replacement to keep constant residue from the DDD of different proteins and the conservative amino acid residue of AD sequence camber, yet not too the residue of high conservative can be changed to produce the AD that describes among this paper and/or the sequence variants of DDD sequence more easily.
Except the sequence variants of DDD and/or AD part, in certain embodiments, can preferably calling sequence variation in the connector peptide sequence of antibody moiety or connection antibody and AD sequence.In an illustrative instance, below 3 possibility variants of fusion rotein sequence are shown in.
(L)
QKSLSLSPGLGSGGGGSGGCG(SEQ?ID?NO：60)
(A)
QKSLSLSPGAGSGGGGSGGCG(SEQ?ID?NO：61)
(-)
QKSLSLSPGGSGGGGSGGCG(SEQ?ID?NO：62)
Amino acid replacement
In certain embodiments, disclosed method and composition can comprise the protein with one or more alternate amino acid residues or the generation and the use of peptide.Can be through replacing structure, physics and/or the treatment characteristic that one or more amino acid residues come optimization natural antibody, chimeric antibody, humanized antibody or people's antibody or AD or DDD sequence.For example, as everyone knows can be through improve the functional character of humanized antibody with a limited number of people's framework region of corresponding FR amino acid replacement (FR) aminoacid of parental generation murine antibody.When framework region amino acid residue and CDR residue very near the time, this situation is especially correct.
In other cases; Can be through the treatment characteristic of a limited number of amino acid replacement optimization antibody, for example to the disassociation of binding affinity, antibody and its target antigen of target antigen or dissociation rate or even antibody to the inductive efficient of CDC (complement dependent cytotoxicity) or ADCC (ADCC).
In selectable embodiment, further optimization is used to produce the DDD and/or the AD sequence of DNL construct of the present invention, for example to increase the DDD-AD binding affinity.Discussed the potential sequence variations in DDD or the AD sequence in the preceding text.
Those skilled in the art will appreciate that generally speaking amino acid replacement generally includes with the another kind of amino acid replacement aminoacid with similar relatively character (that is, conserved amino acid substitutes).The character of different aminoacids and amino acid replacement are the theme of broad research and knowledge in this area to the influence of protein structure and function all the time.
For example, but the hydrophilic index of considered amino acid (Kyte & Doolittle, 1982, J.Mol.Biol., 157:105-132).Amino acid whose hydrophilic relatively characteristic has been facilitated the proteinic secondary structure of gained, and this has confirmed the interaction of protein and other molecule conversely.Each is amino acid based to be endowed hydrophilic index (Kyte & Doolittle, 1982) in its hydrophobicity and charge characteristic, and these aminoacid are: isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine/cystine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (0.4); Threonine (0.7); Serine (0.8); Tryptophan (0.9); Tyrosine (1.3); Proline (1.6); Histidine (3.2); Glutamic acid (3.5); Glutamine (3.5); Aspartic acid (3.5); Agedoite (3.5); Lysine (3.9); And arginine (4.5).In producing conservative substituting, the amino acid whose use of its hydrophilic index ± 2 in is preferably, and its hydrophilic index is preferred in ± 1, and its hydrophilic index is further preferred in ± 0.5.
But amino acid replacement is the hydrophilic of considered amino acid residue (for example, the 4th, 554, No. 101 United States Patent (USP)s) also.Hydrophilicity value has been endowed to amino acid residue: arginine (+3.0); Lysine (+3.0); Aspartic acid (+3.0); Glutamic acid (+3.0); Serine (+0.3); Agedoite (+0.2); Glutamine (+0.2); Glycine (0); Threonine (0.4); Proline (0.5.+-.1); Alanine (0.5); Histidine (0.5); Cysteine (1.0); Methionine (1.3); Valine (1.5); Leucine (1.8); Isoleucine (1.8); Tyrosine (2.3); Phenylalanine (2.5); Tryptophan (3.4).To substitute aminoacid be preferred with having similar hydrophilic other aminoacid.
Other consideration comprises the size of amino acid side chain.For example, for example tryptophan or tyrosine substitute the aminoacid with compact side chain for example glycine or serine are not preferred usually with the aminoacid with bulky side chain.Also to consider of the influence of different aminoacids residue to secondary protein structure.Through empirical studies, the different aminoacids residue to protein domain take the influence of the tendency of alpha-helix, beta-pleated sheet or counter-rotating secondary structure to be determined and to be in this area to be known (referring to, for example; 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 alternate expression of conserved amino acid has been fabricated and has been known in this area.For example, arginine and lysine; Glutamic acid and aspartic acid; Serine and threonine; Glutamine and agedoite; And valine, 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 whether residue is positioned at proteinic inside or is not exposed to solvent.For inner residue, 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. (referring to, for example, the PROWL website that rockefeller.edu is last).For the residue that is 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.).Made up multiple matrix and helped select amino acid replacement, for example 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 (ditto).
In measuring amino acid replacement; Also can consider the existence of intermolecular or intramolecular bond; The formation of disulfide bond between the formation of ionic bond or near the cysteine residues between for example positively charged residue (for example, His, Arg, Lys) and the electronegative residue (for example, Asp, Glu).
With any other the amino acid whose method in any amino acid replacement encoded protein matter sequence is known and normal experiment only to those skilled in the art; For example, it is spliced into the expression vector establishment body carry out then through the technology of direct mutagenesis or through the synthetic and alternate oligonucleotide of assembling coded amino acid.
Therapeutic agent
In selectable embodiment; Can use therapeutic agent; It is conjugated to immunotoxin of the present invention; Or before immunotoxin is used, simultaneously or use it afterwards dividually, said therapeutic agent is for example cytotoxic agent, anti--angiogenic agent, short apoptosis agent, antibiotic, hormone, hormone antagonist, chemotactic factor, medicine, prodrug, toxin, enzyme or other reagent.The medicine that uses can have the medicinal property that is selected from antimitotic agent, antikinase, alkylating agent, antimetabolite, antibiotic, alkaloid, anti--angiogenic agent, short apoptosis agent and its combination.
The illustrative drug of using can comprise 5-fluorouracil, aplidin, azaribine, Anastrozole, anthracene nucleus class, bendamustine, bleomycin, bortezomib, bryostatin 1, busulfan, calicheamicin, camptothecine, carboplatin, 10-hydroxycamptothecine, carmustine, celecoxib, chlorambucil, cisplatin (CDDP), cox 2 inhibitor, irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecine, cyclophosphamide, cytosine arabinoside, dacarbazine, docetaxel, actinomycin D, daunorubicin, doxorubicin, 2-pyrrolin doxorubicin (2P-DOX), cyanic acid-morpholino doxorubicin, glucosiduronic acid doxorubicin, glucosiduronic acid epirubicin, estramustine, epipodophyllotoxin, estrogen receptor bonding agent, etoposide (VP16), glucosiduronic acid etoposide, phosphoric acid etoposide, floxuridine (FUdR), 3 ', 5 '-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, farnesyl-protein converting enzyme inhibitor, gemcitabine, hydroxyurea, idarubicin, ifosfamide, L-Asnase, lenalidomide, calcium folinate, lomustine, chlormethine, melphalan, mercaptopurine, Ismipur, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, nvelbine, nitrourea, plicamycin (plicomycin), procarbazine, paclitaxel, pentostatin, PSI-341, raloxifene, semustine, streptozocin (streptozocin), tamoxifen, taxol, temozolomide's (moisture form of DTIC), anti-platinum (transplatinum), Thalidomide, thioguanine, plug be for group, teniposide, hycamtin, uracil mustard, vinorelbine, vinblastine, vincristine and vinca alkaloids.
The toxin that uses can comprise Ricin, abrin, alpha toxin, saporin, ribonuclease (RNA enzyme), for example ranpirnase, DNA enzyme I, SEA, pokeweed antiviral protein, gelonin, diphtheria toxin, diphtherotoxin, PE and pseudomonas endotoxin.
The chemotactic factor that uses can comprise RANTES, MCAF, MIP1-α, MIP1-β and IP-10.
In certain embodiments; Can use for example angiostatin (angiostatin) of anti--angiogenic agent, baculostatin, blood vessel can chalones (canstatin), the mammary gland silk presses down albumen, 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-inhibitive factor) antibody, laminin peptide, fibronectin peptide, PAI, tissue inhibitor of metalloproteinase, interferon, interleukin 12, IP-10, Gro-β, thrombospondin, 2-methoxyestradiol, proliferin GAP-associated protein GAP, carboxylic acid amides triazole (carboxiamidotriazole), CM101, Marimastat (Marimastat), the many sulfate of pentosan, angiopoietin-2, interferon-ALPHA, Antibiotic TAN 420F, PNU145156E, 16K prolactin antagonist fragment, Linomide (linomide), Thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, accutin, angiostatin, GS-504, vincristine, bleomycin, AGM-1470, platelet factor 4 or minocycline.
The immunomodulator that uses can be selected from cytokine, stem cell factor, lymphotoxin, Hemopoietic factor, colony stimulating factor (CSF), interferon (IFN), erythropoietin, thrombopoietin and its combination.Useful especially be lymphotoxin for example tumor necrosis factor (TNF), Hemopoietic factor for example interleukin (IL), colony stimulating factor for example granulocyte-colony stimulating factor (G-CSF) or CM-CSF (GM-CSF), interferon for example interferon-' alpha ' ,-β or-γ and the stem cell factor stem cell factor of called after " the S1 factor " for example.Comprise growth hormone for example human growth hormone, N-methionyl human growth hormone and BGH in the cytokine; Parathyroid hormone; Thyroxine; Insulin; Proinsulin; Relaxin; Relaxation precipitinogen; Glycoprotein hormones is FSH (FSH), thyroid-stimulating hormone (TSH) and lutropin (LH) for example; Hepatocyte growth factor; Prostaglandin, fibroblast growth factor; Prolactin antagonist; Galactagogin, OB albumen; Tumor necrosis factor-alpha and-β; Mullerian inhibiting substance (mullerian inhibiting substance); Mus GTH related peptides; Inhibin; Activin; VEGF; Integrate plain; Thrombopoietin (TPO); Nerve growth factor is NGF-β for example; PDGF; Transforming growth factor (TGF) is TGF-α and TGF-β for example; Insulin like growth factor-1 and-II; Erythropoietin (EP0); Bone-inducing factor; Interferon for example interferon-' alpha ' ,-β and-γ; Colony stimulating factor (CSF) is macrophage-CSF (M-CSF) for example; Interleukin (IL) is 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 for example; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit part or FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factor and LT.
The radionuclide that uses 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 with 211Pb.Therapeutic radiation property nucleic preferably has 60 to 6, the decay ability in the 000keV scope, and the decay of Auger emitter can be preferably in 60 to the 200keV scopes; 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, and 000keV more preferably is 100-4, and 000keV is most preferably 500-2,500keV.Basically the radionuclide that decays through radiation Auger particle also is preferred.For example, 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＜and 1,000keV more preferably is＜100keV to be most preferably＜70keV.Basically the radionuclide that decays through the generation alpha-particle also is 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, and 000keV more preferably is 3,000-8,000keV is most preferably 4,000-7,000keV.Other the potential radiosiotope that uses 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.
Therapeutic agent can comprise photosensitizer or dyestuff.Used fluorescent composition for example fluorescent dye and other chromophore or dyestuff (for example to visible responsive porphyrin) detect and treat pathological changes, promptly use suitable rayed diseased region.In treatment, this is called as light radiation, phototherapy or PDT.Referring to people such as Jori (volume), PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES (Libreria Progetto 1985); Van den Bergh, Chem.Britain (1986), 22:430.In addition, with monoclonal antibody and light-sensitive coloring agent coupling, be used to carry out phototherapy.Referring to people such as Mew, J.Immunol. (1983), 130:1473; Idem., Cancer Res. (1985), 45:4380; People such as Oseroff, Proc.Natl.Acad.Sci.USA (1986), 83:8744; Idem., Photochem.Photobiol. (1987), 46:83;
Other useful therapeutic agent can comprise oligonucleotide, and the for example antisense oligonucleotide of bcl-2 or p53 of antioncogene and oncoprotein particularly preferably leads.The preferred form of therapeutic oligonucleotide is siRNA.
Diagnostic agent
Diagnostic agent is preferably selected from radionuclide, radiocontrast medium, paramagnetic ion, metal, fluorescent marker, chemiluminescent labels, acoustic contrast agent and photosensitive reagents.This type of diagnostic agent is known and can uses any this type of known diagnostic agent.The non-limiting example of diagnostic agent for example can comprise radionuclide 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 that uses comprises chromium (III), manganese (II), ferrum (III), ferrum (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 for example liposome of gas filling of liposome.Radiopaque diagnostic agent can be selected from chemical compound, barium compound, gallium compound and thallium compound.Many kinds of fluorescent markers are known in this area, include but not limited to Fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, phthalic aldehyde and fluorescamine.The chemiluminescent labels that uses can comprise luminol, different luminol, aromatic series acridinium ester, imidazoles, acridinium salt or oxalate.
The method of therapeutic treatment
Different embodiments relates to the for example method for cancer of mammal (comprise the people, raise and train or companion pet for example Canis familiaris L. and cat) of treatment experimenter, comprises the cytotoxic immune conjugate to said experimenter's administering therapeutic effective dose.
In one embodiment, the immunological diseases of immunotoxin of the present invention treatment capable of using for example can comprise joint disease for example ankylosing spondylitis, juvenile rheumatoid arthritis, rheumatoid arthritis; Sacred disease is multiple sclerosis and myasthenia gravis for example; Pancreatic diseases is diabetes for example, particularly juvenile onset diabetes; Gastroenteropathy is chronic active hepatitis, celiac disease, ulcerative colitis, Crohn disease, pernicious anemia for example; Dermatosis is psoriasis or scleroderma for example; Allergic disease for example asthma with transplant associated conditions for example graft versus host disease and allograft rejection.
Can replenish using of cytotoxic immune conjugate through the another kind of antibody of administering therapeutic effective dose simultaneously or one after the other (the lip-deep another kind of antigen of said antibodies target cell or with said antigen-reactive).Preferred other MAb comprises at least a humanization MAb, chimeric MAb or people MAb, and it is selected from MAb:CD4, CD5, CD8, CD14, CD15, CD16, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD70, CD74, CD79a, CD80, CD95, CD126, CD133, CD138, CD154, CEACAM5, CEACAM6, B7, AFP, PSMA, EGP-1, EGP-2, carbonic anhydrase IX, PAM4 antigen, MUC1, MUC2, MUC3, MUC4, 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 with following substance reaction.For example anti--CD19, anti-CD 20 and anti--CD22 antibody is known to the different antibodies of using to those skilled in the art.Referring to, for example, people such as Ghetie, Cancer Res.48:2610 (1988); People such as Hekman, Cancer Immunol.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; 7,312,318; 7,501,498; 7,612,180; 7,670, No. 804 United States Patent (USP)s; With the 20080131363rd; 20070172920; 20060193865 and No. 20080138333 U.S. Patent Application Publication are incorporated the embodiment part of above-mentioned each piece into this paper by reference.
Also can or one after the other use at least a therapeutic agent and come further to replenish the immunotoxin therapy through the while." CVB " (15g/m for example 2Cyclophosphamide, 200-400mg/m 2Etoposide and 150-200mg/m 2Carmustine) is the scheme that is used to treat non--He Jiejin lymphomas.People such as Patti, Eur.J.Haematol.51:18 (1993).Other suitable associating chemistry therapeutic scheme is known to those skilled in the art.Referring to, for example, people such as Freedman, " Non-Hodgkin ' s lymphomas, " in CANCER MEDICINE, the 2nd volume, the 3rd edition, people such as Holland (volume), 2028-2068 page or leaf (Lea & Febiger 1993).As illustrating, the first generation chemotherapy scheme that is used to treat moderate non--He Jiejin lymphomas (NHL) comprises C-MOPP (cyclophosphamide, vincristine, procarbazine and prednisone) and CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone).Useful second filial generation chemotherapy scheme is m-BACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone and a folinic acid), and suitable third generation scheme is MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone, bleomycin and a folinic acid).Other useful medicine comprises phenylbutyric acid salt, bendamustine and bryostatin-1.
Can prepare the compositions of immunotoxin of the present invention by known method, by immunotoxin and pharmaceutically suitable vehicle group are combined in the mixture with the preparation pharmaceutically useful.Aseptic phosphate buffer is an instance of pharmaceutically suitable excipient.Other appropriate excipients is known to those skilled in the art.Referring to, for example, people such as Ansel; PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS (pharmaceutical dosage form and drug delivery system); The 5th edition (Lea Febiger 1990), and Gennaro (volume), REMINGTON ' S PHARMACEUTICAL SCIENCES (Lei Mingdunshi pharmaceutical science); The 18th edition (Mack Publishing Company 1990), and revision version.
Can immunotoxin preparation of the present invention be used for intravenous administration, through for example bolus infusion or continuous infusion.Preferably, be shorter than in about 4 hours period, more preferably be shorter than infusion immunotoxin in about 3 hours period.For example, can be in 30 minutes, preferred in addition 15 minutes in 25-50mg before the infusion, infusion remainder in subsequently 2-3 hour.Injection preparation can be unit dosage forms, for example is contained in the ampoule, perhaps is contained in the multi-dose container and the interpolation antiseptic.Compositions can adopt the for example form of the suspension in oiliness or aqueous vehicles, solution or emulsion, and can contain formula agent (formulatory agents) for example suspending agent, stabilizing agent and/or dispersant.Selectively, active component can be powder form, is used for using before use suitable vehicle (for example aseptic apirogen water) preparation.
Can other pharmaceutical methods be used to control the acting duration of immunotoxin.Controlled release preparation can be realized through using the biocompatible polymer complexation or adsorbing immunotoxin.For example, biocompatible polymer comprises the polyanhydride copolymer substrate of ethylene-vinyl acetate copolymer substrate and stearic acid dimer and decanedioic acid.People such as Sherwood, Bio/Technology 10:1446 (1992).From then on the speed that discharges on the substrate depends on the amount of the molecular weight of this immunotoxin, intramatrical immunotoxin and the size of dispersed particle.People such as Saltzman, Biophys.J.55:163 (1989); People such as Sherwood, the same.Other solid dosage forms is described in people such as Ansel; PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS (pharmaceutical dosage form and drug delivery system); The 5th edition (Lea Febiger 1990) and Gennaro compile, REMINGTON ' S PHARMACEUTICAL SCIENCES (Lei Mingdunshi pharmaceutical science); The 18th edition (Mack Publishing Company1990), and revision version.
Also can be subcutaneous or even be applied to mammal through other parenteral approach with immunotoxin.In addition, can inject through continuous infusion or through single or multiple and use.Preferably, be shorter than in about 4 hours period, more preferably be shorter than infusion immunotoxin in about 3 hours period.
More common, the application dosage that is used for people's immunotoxin depends on such as factors such as patient age, body weight, height, sex, general health and previous treatment history and changes.Maybe be to the dosage of the immunotoxin in the receptor provides about 1mg/kg to 25mg/kg scope as the single intravenous infusion, but also can according to circumstances need use lower or higher dosage.The dosage of using by 1-20mg/kg for 70kg patient for example is 70-1,400mg, or for 1.7 meters patient, dosage is 41-824mg/m 2Can repeat this dosage on demand, for example weekly, continue 4-10 week, or weekly, continue 8 weeks or weekly, continued for 4 weeks.Frequency administration that can also be lower for example week about once, continues the several months, or every month once or per season once, continue many months, as required in the maintenance therapy.
Selectively, with per 2 or 3 Monday a dosage use immunotoxin, repeat at least 3 dosage altogether.Perhaps, the said construct of administered twice continues 4-6 week weekly.If dosage is reduced to about 200-300mg/m 2(each dosage 340mg (for 1.7 meters patient) or 4.9mg/kg (for the patient of 70kg), can use weekly once or even secondary, continued for 4 to 10 weeks.Selectively, can reduce dosage schedule, promptly per 2 or 3 weeks once continued 2-3 month.Yet, confirmed even can use higher dosage (for example weekly or every 2-3 week 20mg/kg) through intravenous infusion slowly, carry out multiple administration circulation.Can be randomly with other repeat administration scheme at interval, and can dosage be provided through different parenteral approach (dosage and arrangement of time are carried out suitable adjustment).
In preferred embodiments, immunotoxin is used for treatment for cancer.The instance of cancer includes but not limited to cancer, lymphoma, glioblastoma multiforme, melanoma, sarcoma and leukemia, myeloma or lymph appearance malignant disease tumor (lymphoid malignancies).Hereinafter point out the instance more specifically of this type of cancer; Comprise: squamous cell carcinoma (for example, epithelium squamous cell carcinoma), Ewing sarcoma, Wilms tumor, astrocytoma, pulmonary carcinoma comprise that squamous cell carcinoma, peritoneal cancer, hepatocarcinoma, human primary gastrointestinal cancers (gastric cancer) or the gastric cancer of adenocarcinoma and the lung of small cell lung cancer, nonsmall-cell lung cancer, lung comprise thyroid carcinoma, breast carcinoma, ovarian cancer, colon cancer, rectal cancer, endometrium cancer or uterus carcinoma, salivary-gland carcinoma, renal carcinoma or renal carcinoma (renal cancer), carcinoma of prostate, carcinoma vulvae, anus cancer, carcinoma of penis and the head and neck cancer of human primary gastrointestinal cancers, cancer of pancreas, glioblastoma multiforme, cervical cancer, ovarian cancer, hepatocarcinoma, bladder cancer, hepatoma, hepatocarcinoma, NET, medullary thyroid carcinoma (medullary thyroid cancer), differentiation.Term " cancer " (for example comprises primary malignancy cell or tumor; Its cell does not also migrate to the malignant cell or the tumor of position of the experimenter's except the position of original malignant tumor or tumor health) and Secondary cases malignant cell or tumor (for example, through the transfer of malignant cell or tumor cell to the Secondary cases position different with the position of original tumor, malignant cell or the tumor that migration produces).The cancer that is suitable for Therapeutic Method of the present invention comprises expression, crosses the cell of expression or unconventionality expression IGF-1R.
Other examples of cancer or cancer include, but are not limited to: children with acute lymphoblastic leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia (Acute? Lymphocytic? Leukemia), acute myeloid leukemia, adrenocortical carcinoma, adult (primary sex) carcinoma, adult (primary) liver cancer, adult acute lymphoblastic leukemia, adult acute myeloid leukemia, adult Hodgkin's lymphoma, adult lymphoblastic leukemia, adult non - Hodgkin lymphoma, adult primary liver cancer, adult soft tissue sarcoma, AIDS-related lymphoma, AIDS-related cancer, colorectal cancer, astrocytoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumors, breast cancer, renal pelvis and ureter cancer, primary central nervous system lymphoma, central nervous system lymphoma, cerebellar astrocytoma (Cerebellar? Astrocytoma), astrocytoma (Cerebral? Astrocytoma), cervical cancer, children ( primary) hepatocellular carcinoma, children (primary) liver cancer, childhood acute lymphoblastic leukemia (Childhood? Acute? Lymphoblastic? Leukemia), children with acute myeloid leukemia, children's brain stem glioma, cerebellar child star cell tumor (Childhood? Cerebellar? Astrocytoma), Children cerebellar astrocytoma Childhood? Cerebral? Astrocytoma), children extracranial germ cell tumors (Childhood? Extracranial? Germ? Cell? Tumors), Children Hodgkin (Childhood? Hodgkin's? Disease), children Tong Hejie lymphoma, children hypothalamus and visual pathway glioma, children lymphocytic leukemia (Childhood? Lymphoblastic? Leukemia), children with medulloblastoma (Childhood? Medulloblastoma), children Non - Hodgkin's lymphoma, a children's pineal and supratentorial primitive neuroectodermal tumor, children with primary liver cancer, rhabdomyosarcoma children, children soft tissue sarcoma, a children's visual pathway and hypothalamic glioma, chronic lymphocytic leukemia, chronic myeloid leukemia, colon cancer, cutaneous T-cell lymphoma, endocrine islet cell cancer, endometrial cancer, ependymoma, epithelial cancer, esophageal cancer, Ewing sarcoma and related tumors, exocrine pancreatic cancer, extracranial blastoma (Extracranial? Germ? Cell? Tumor), extragonadal germ cell tumors (Extragonadal? Germ? Cell? Tumor), extrahepatic bile duct cancer, eye cancer, female breast cancer, Gaucher disease (Gaucher's? Disease ), gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors (Gastrointestinal? Carcinoid? Tumor), gastrointestinal tumors (Gastrointestinal? Tumors), germ cell tumors, gestational trophoblastic tumor, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, HE Hodgkin's lymphoma, high agammaglobulinemia (Hypergammaglobulinemia), hypopharyngeal cancer, colon cancer, intraocular melanoma, pancreatic islet cell carcinoma, pancreatic islet cells, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cavity cancer, liver cancer, lung cancer, lymphoproliferative disorders (Lymphoproliferative? Disorders), macroglobulinemia, male breast cancer, malignant mesothelioma (Malignant? Mesothelioma), malignant thymoma, medulloblastoma, Black melanoma, mesothelioma, metastatic potential of primary squamous cervical cancer (Metastatic? Occult? Primary? Squamous? Neck? Cancer), metastatic primary squamous cervical cancer (Metastatic? Primary? Squamous? Neck? Cancer), metastatic squamous neck cancer (Metastatic? Squamous? Neck? Cancer), multiple myeloma, multiple myeloma / plasma cell neoplasms, myelodysplastic syndrome, myeloid leukemia (Myelogenous? Leukemia), medullary leukemia (Myeloid? Leukemia), myeloproliferative disorders, nasal and sinus cancer (Nasal? Cavity? and? Paranasal? Sinus? Cancer), nasopharyngeal cancer, neuroblastoma, non - Hodgkin's lymphoma, non-black melanoma skin cancer, non-small cell lung cancer, metastatic occult primary squamous cervical carcinoma (Occult? Primary? Metastatic? Squamous? Neck? Cancer), oropharyngeal cancer, osteosarcoma / malignant fibrosarcoma, osteosarcoma / malignant fibrous histiocytoma, osteosarcoma / malignant fibrous histiocytoma of bone, epithelial ovarian cancer, ovarian germ cell tumors, ovarian low malignant potential tumors (Ovarian? Low? Malignant? Potential? Tumor), pancreatic cancer, paraproteinemia (Paraproteinemias), polycythemia vera, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumors, primary central nervous system lymphoma (Primary? Central? Nervous? System? Lymphoma), primary liver cancer, prostate cancer, colorectal cancer, renal cell carcinoma, renal pelvis and ureter cancer (Renal? Pelvis? and? Ureter? Cancer), retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sarcoidosis sarcoma, race zari's syndrome (Sezary? Syndrome), skin cancer, small cell lung cancer, small bowel cancer, soft tissue sarcoma, squamous cervical carcinoma (Squamous? Neck? Cancer), gastric cancer, supratentorial primitive neuroectodermal and pineal tumors, T-cell lymphoma, testicular cancer, thymoma, thyroid cancer, renal pelvis and ureter transitional cell carcinoma (Transitional? Cell? Cancer? of? the? Renal? Pelvis? and? Ureter), renal pelvis and ureter transitional cell carcinoma (Transitional? Renal? Pelvis? and? Ureter? Cancer), trophoblastic tumor (Trophoblastic? Tumors), ureter and renal pelvis carcinoma (Ureter? and? Renal? Pelvis? Cell? Cancer), urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, visual pathways and hypothalamus glioma, vulvar, Walden Manchester macroglobulinemia, Wilms tumor, and any other hyperproliferative disease, besides neoplasia formation, but are located in the organ systems listed above.
The method and composition of describing among this paper with requiring to protect can be used for treating pernicious or worsens preceding state and be used to stop the progress to neoplasia state (neoplastic state) or malignant state (including but not limited to above-mentioned those obstacles).This type of purposes is applicable to known or suspects and proceeds to neoplasia or cancer; Particularly wherein by hyperplasia, change to give birth to (metaplasia) or the most especially the non-neoplasia cell formed of dysplasia grow the disease that taken place (about the summary of this type of misgrowth condition, referring to Robbins and Angell, Basic Pathology; The 2nd edition; W.B.Saunders Co., Philadelphia, pp.68-79 (1976)) the state of progress.
Dysplasia is the tendency of cancer normally, and is mainly seen in the epithelium.It is the most disorderly form of non-neoplasia cell growth, comprises the loss of the structure of individual cells concordance and cell towards (architectural orientation).When having chronic stimulation or inflammation, dysplasia characteristic ground takes place.The dysplasia obstacle that can be treated includes but not limited to anhidrotic ectodermal dysplasia (anhidrotic ectodermal dysplasia); Heteropleural dysplasia (anterofacial dysplasia); Asphyxiating thoracic dysplasia; Atriodigital dysplasia (atriodigital dysplasia); Broncho-pulmonary dysplasia; Cerebral dysplasia (cerebral dysplasia); The cervix uteri dysplasia; Chondroectodermal dysplasia; CCD; Congenital ectodermal dysplasia; Craniodiaphyseal dysplasia (craniodiaphysial dysplasia); Cranium wrist metatarsal hypoplasia (craniocarpotarsal dysplasia); Skull metaphysis hypoplasia (craniometaphysial dysplasia); Dentinal dysplasia (dentin dysplasia); Camurati-Engelmann disease (diaphysial dysplasia); Ectodermal dysplasia (ectodermal dysplasia); Enamel hypoplasia (enamel dysplasia); Encephalo-ophthalmic dysplasia (encephalo-ophthalmic dysplasia); Dysplasia epiphysialis hemimelia (dysplasia epiphysialis hemimelia); Multiple epiphyseal dysplasia (dysplasia epiphysialis multiplex); Chondrodystrophia congenita punctata (dysplasia epiphysialis punctata); Epithelial dysplasia (epithelial dysplasia); Face-refer to (toe)-genital development bad (faciodigitogenital dysplasia); Familial fibrous dysplasia of jaw (familial fibrous dysplasia of jaws); Familial white pleat abnormal development (familial whitefolded dysplasia); Fibromuscular dysplasia (fibromuscular dysplasia); Fibrous dysplasia (fibrous dysplasia of bone); Florid osseous dysplasia (florid osseous dysplasia); Heritability kidney-retinal dysplasia (hereditary renal-retinal dysplasia); Perspiration property is ectodermal dysplasia (hidrotic ectodermal dysplasia); Hypohidrotic ectodermal dysplasia (hypohidrotic ectodermal dysplasia); Lymphopenia property thymic dysplasia (lymphopenic thymic dysplasia); Breast dysplasia (mammary dysplasia); Following maxillofacial development bad (mandibulofacial dysplasia); Metaphysis dysplasia (metaphysial dysplasia); Cover end Buddhist nun abnormal development (Mondini dysplasia); Single fibrous dysplasia of bone (monostotic fibrous dysplasia); Mucous epithelium dysplasia (mucoepithelial dysplasia); Multiple epiphyseal dysplasia (multiple epiphysial dysplasia); Eye-Er-vertebra syndrome (oculoauriculovertebral dysplasia); Oculo-dental-digital (toe) dysplasia (oculodentodigital dysplasia); Oculovertebral dysplasia (oculovertebral dysplasia); Odontodysplasia (odontogenic dysplasia); Opthalmomandibulomelic dysplasia; Periapical cementum dysplasia (periapical cemental dysplasia); Polyostotic fibrous dysplasia (polyostotic fibrous dysplasia); Pseudoachondroplasia property spondylo epiphyseal dysplasia (pseudoachondroplastic spondyloepiphysial dysplasia); Retinal dysplasia (retinal dysplasia); Separated-hypoplasia of optic nerve (septo-optic dysplasia); Vertebra aplasia of epiphysis (spondyloepiphysial dysplasia) and ventriculoradial dysplasia.
Obstacle includes but not limited to optimum hyperplasia sexual disorders (for example, benign tumor, fibrous capsule sexual state, tissue hypertrophy, polyp intestinal or adenoma and esophagus dysplasia), white macula, keratosis, bowen, farmer's skin sick (Farmer ' s Skin), solar cheilitis and solar keratosis before the other tumor that can be treated.
In preferred embodiments, method of the present invention is used to suppress cancer particularly growth, progress and/or the transfer of listed those cancers of preceding text.
Other excess proliferative disease, obstacle and/or state include but not limited to the progress and/or the transfer of malignant tumor and associated disorders; Said malignant tumor and associated disorders are that for example leukemia (comprises that acute leukemia (for example; Acute lymphoblastic leukemia, acute myelocytic leukemia (comprising myeloblastic leukemia), promyelocytic leukemia), the bone marrow mononuclear cell leukemia), monocytic leukemia) and erythroleukemia)) and chronic leukemia (for example; Chronic myelocytic (granulocyte) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (for example; Hokdkin disease and Fei Hejiejinshi are sick), multiple myeloma, Walden Si Telun macroglobulinemia, heavy chain disease and solid tumor, include but not limited to sarcoma and cancer for example fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, Ewing sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, cancer of pancreas, breast carcinoma, ovarian cancer, carcinoma of prostate, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, syringocarcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchiogenic cancer, renal cell carcinoma, hepatoma (hepatoma), cancer of biliary duct, choriocarcinoma, spermocytoma, embryonal carcinoma, nephroblastoma ((Wilm ' s tumor), cervical cancer, tumor of testis, pulmonary carcinoma, small cell lung cancer, bladder cancer, cell carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma (emangioblastoma), acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma.
Expression vector
Other embodiment can relate to and comprise the for example DNA sequence of the nucleic acid of antibody, antibody fragment, toxin or the composition fusion rotein of DNL construct of coding immunotoxin.Fusion rotein can comprise antibody or fragment or the toxin that is connected to AD for example or DDD part.
Various embodiments relate to the expression vector that comprises DNA sequences encoding.Said carrier can comprise the sequence of light chain and the CH and the hinge region of coding human immunoglobulin, can this sequence be connected with chimeric, humanization or people's variable region sequences.Said carrier can be included in promoter, enhancer and signal sequence or the targeting sequencing of expressing encoded protein matter in the selected host cell extraly.Useful especially carrier is pdHL2 or GS.More preferably; Light chain and CH and hinge region can be from people EU myeloma immunoglobulins; Wherein randomly with on the allotype position amino acid whose at least one change over the aminoacid of in different I gG1 allotype, finding, and wherein randomly available alanine substitutes the aminoacid 253 based on the heavy chain of the EU of EU numbering system.Referring to people such as Edelman, Proc.Natl.Acad.Sci USA 63:78-85 (1969).In other embodiments, can the IgG1 sequence be transformed into the IgG4 sequence.
It will be understood by those skilled in the art that genetic engineering modified expression vector and insert host cell and be known in the art and normal experiment only with the method for expressing engineered protein.Antibody or segmental host cell and the method in for example the 7th, 531, No. 327 and the 7th, 537, No. 930 United States Patent (USP)s (its embodiment part is separately incorporated this paper by reference into), having described to be used for expression cloning.
Test kit
Various embodiments can relate to containing and are suitable for treating or the test kit of the component of the illing tissue of diagnosing patients.Exemplary kit can comprise the immunotoxin of describing among one or more this paper.Do not process through digestive tract (sending like administered through oral) and send if comprise the compositions of using component, then also can comprise can be through the equipment of some other approach delivery of agents box components for test kit.A kind of to be used to use the device type that parenteral for example sends be syringe, and it is used for said compositions is expelled in the subject.Also can use inhalation device.In certain embodiments, can with prefilled syringe that sterile liquid formulations or lyophilized formulations are housed or automatically the form of injection pen (autoinjection pen) therapeutic agent is provided.
Said reagent constituents can packaging together or separately be packaged in two or more containers.In certain embodiments, said container can be the phial that comprises aseptic, the lyophilized formulations of the compositions of be fit to rebuilding (reconstitution).Test kit also can comprise one or more buffer that are applicable to the reconstruction and/or dilute other reagent.Other spendable container includes but not limited to: bag, dish, box, pipe etc.But the reagent constituents aseptic packaging also is kept in the said container.Another ingredient that can comprise is for giving the personnel's that use test kit operation instruction.
Embodiment
The following example is provided in order to illustrating, but does not limit claim of the present invention.
Embodiment 1. is had the effective cell toxicity of anti-different people epithelial cancer cells by the ranpirnase (Frog RNase) of humanization, internalization, anti--Trop-2 antibody target
Show among this paper comprising reorganization be bound to humanization anti--the novel immunotoxin of the Amphibian ribonuclease of Trop-2 antibody shows the extensive and strong antiproliferative activity of anti-people's pulmonary carcinoma xenograft in external anti-various human epithelial cancer cell line (for example cervical cancer, breast carcinoma, colon cancer, cancer of pancreas, ovarian cancer and carcinoma of prostate) and the body.
Summary
We disclose the novel immunotoxin based on IgG of called after 2L-Rap (Q)-hRS7 in this article, and it comprises Rap (Q) (removing the mutant form of Rap of the N glycosylation site of supposition) and hRS7 (the internalization humanized antibody of anti-Trop-2 (in multiple epithelial cancer, crossing the cell surface glycoprotein of expressing)).Different tests, comprise size exclusion HPLC, SDS-PAGE, flow cytometry, RNA enzymatic activity, internalization, cell viablity (cell viability) and colony form the purity of proving this immunotoxin, molecule integrity, to the comparable affinity of the hRS7 of the cell line that combines several kinds of different expression Trop-2 and the effectiveness of the growth of these cell lines of inhibition on nanomolar concentration.2L-Rap (Q)-hRS7 also suppressed tumor growth in lotus has the prophylaxis model of nude mouse of Calu-3 people's nonsmall-cell lung cancer xenograft, the meta time-to-live (MST) increased to 96 days (P＜0.01) from 55 days.These results have proved that 2L-Rap (Q)-hRS7 is as the cancer of the multiple expression Trop-2 good effect of cervical cancer, breast carcinoma, colon cancer, cancer of pancreas, ovarian cancer and treatment of prostate cancer agent for example.
Materials and methods
Cell line and cell cultureCervical cancer strain (ME-180), breast carcinoma strain (T-47D, MDA-MB-468, SK-BR-3), carcinoma of prostate strain (DU-145, PC-3,22Rv1), adenocarcinoma of lung strain (Calu-3), cancer of pancreas strain (Capan-1, BxPC-3 and AsPc-1) and ovarian cancer strain (SK-OV-3) are available from American type culture collection (Manassas; VA), and with it at 37 ℃, 5%CO 2Be incubated at down in the RPMI1640 culture medium that is supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2mM L-glutaminate, 200 unit/mL penicillins and 100 μ g/mL streptomycins.
Antibody and reagentMilatuzumab (hLL1, anti--CD74), hRS7, reorganization ranpirnase (rRap) and mouse anti-Rap IgG be from Immunomedics.Fluorescein isothiocyanate (FITC)-, the goat Anti-Human (GAH) of phycoerythrin (PE)-or horseradish peroxidase (HRP)-put together or goat anti-mice (GAM) IgG, Fc-specific antibody available from Jackson ImmunoResearch Labs (West Grove, PA).Be conjugated to the GAH IgG of Alexa Fluor 488, the HTrf who is conjugated to Alexa Fluor 568 and Hoechst 33258 all available from Molecular Probes (Invitrogen, Carlsbad, CA).All restricted enzyme available from New England Biolabs (Beverly, MA).
The structure of carrierThe structure that is used for expressing the plasmid pdHL2-Rap-L-hLL1-γ 4P of 2L-Rap-hLL1-γ 4P is described in hereinafter embodiment 2.Expression vector pdHL2-Rap (Q)-L-hLL1-γ 4P derives from pdHL2-Rap-L-hLL1-γ 4P through substituting Rap with Rap (Q), and plasmid pdHL2-Rap (the Q)-L-hRS7-γ 1 that is used for expressing (Q)-hRS7 is through being able to Rap (Q) gene structure from pdHL2-Rap (Q)-L-hLL1-γ 4P sub-clone to pdHL2-hRS7-γ 1 carrier.In brief, use suitable primer, introduce the EcoRV restriction enzyme site in N-terminal (the 5 ') side of hRS7 VL gene through PCR.The XbaI-EcoRV fragment that will comprise pdHL2-Rap (the Q)-L-hLL1-γ 4P of leader peptide-Rap-connector connects into intermediate carrier pBS-Rap (Q)-L-hRS7 with the EcoRV-BamHI fragment that comprises hRS7 VL gene that produces through PCR.The Xba-BamHI fragment of pdHL2-hRS7-γ 1 replaces with the Xba-BamHI fragment of pBS-Rap (Q)-L-hRS7.
Transfection and selectionPdHL2-Rap (Q)-L-hRS7-γ 1 carrier (30 μ g) carries out linearisation with Sal I; Be transfected into the Sp2/0 cell through electroporation method then, with said cell culture in the complete hybridoma serum-free medium that is supplemented with 10%Low-IgG FBS, 100 unit/mL penicillins and 100 μ g/mL streptomycins, 2mM L-glutaminate, 1 μ M Sodium Pyruvate, 100 μ M essential amino acids and 0.05 μ M methotrexate (MTX).GAH IgG, the Fc-specific antibody of HRP puted together in use, through the Expression of Fusion Protein of elisa assay from the culture supernatant in the hole of survivaling cell.Amplification positive colony and it is freezing to treat use in the future.
Express and purificationWith cell culture extremely final cultivation (viablity of 10-20%) in rolling bottle.With supernatant liquid filtering, be used for previous then with the equilibrated protein A pillar of pH 8.5 buffer that contains 20mM Tris-HCl and 100mMNaCl.After the loading,, use 100mM sodium citrate buffer solution (pH 3.5) to carry out eluting then to obtain fusion rotein with 100-mM sodium citrate buffer solution (pH 7.0) washing pillar.Use 3M Tris-HCl, the peak that pH 8.5 will comprise product is adjusted to pH 7.0, and (PBS) dialyses to the 40mM PBS.After concentrating, product is filtered through 0.22 μ m filter, store down in 2-8 ℃.
Size exclusion HPLC and SDS-PAGE analyzeUse available from BioRad (Hercules, Zorbax pillar CA) be through size exclusion HPLC, and use the 4-20%Tris-glycine gels (
Figure BDA0000139409280000531
Gold Precast Gels, Cambrex, Rockland ME) passes through purity and the molecule integrity that SDS-PAGE assesses (Q)-hRS7 under reductive condition.
In vitro transcription and translation (IVTT) are measuredDescription through according to manufacturer is used
Figure BDA0000139409280000532
(WI) activity of the luciferase of measurement de novo synthesis in cell free system, measures the RNA enzymatic activity for Promega, Madison in Quick Coupled Transcription/Translation system.In brief; To 8 μ L contain add 2 μ L among
Figure BDA0000139409280000533
Quick Master Mix of methionine and luciferase-contrast DNA concentration range in the various specimen between the 10pM to 100nM; With its under 30 ℃ in 96 hole circle base plates incubation 2 hours, from said hole, take out 2 μ L and be used for utilizing 50 μ L Bright-Glo substrates to analyze at black 96 hole flat undersides.On Envision chemiluminescence readings device, read plate.With the concentration mapping of the plain enzyme unit of relative fluorescence (RLU) to specimen.
Yeast tRNA degraded measure also can through measure use yeast tRNA (Invitrogen) as the amount of the perchloric acid solubility nucleotide of substrate formation measure the RNA enzymatic activity (people such as Newton, Blood 2001; 97:528-35).(Ambion, Austin TX) do not have each sample of preparation in the RNA enzyme Eppendorf pipe at 1.5-mL, in the final volume of 100 μ L, to comprise 5nM (Q)-hRS7 or rRap with the water that does not contain the RNA enzyme; 10mM HEPES, pH6.0; 200 μ g/mL human serum albumins; And scope is at the tRNA of 100 μ g/mL (3.09 μ M) to the predetermined concentration between the 600 μ g/mL (18.54 μ M).Under 37 ℃, carry out enzymatic reaction 2 hours, come cessation reaction through the freezing perchloric acid that in each sample, adds 233 μ L 3.4% on ice.After 10 minutes, in the cold house with 12,000rpm centrifugal sample 10 minutes in microcentrifuge.From the supernatant of each sample, take out five equilibrium, 10 times of dilute with waters are blank with water, measure the optical density (OD) from the 260nm place of said diluent.Through the OD value of correspondence is calculated the initial rate of each concentration of substrate divided by the response time (7200 seconds); It is mapped to concentration of substrate; Measuring kcat/Km, according to Mi-Men Er Shi equation (Michaelis-Menten equation) its Km＞＞condition of [S] under, the line of least squares that should equal gained divided by total enzyme concentration (for rRap; 5nM and for (Q)-hRS7, slope 10nM).
Cell combines measurement will be used for the combination of assessment (Q)-hRS7 to selected cell line as follows based on the method for ELISA.Cell is coated on black 96 hole flat undersides (1 * 10 5Individual cells/well; 100 μ L/ holes) and under 37 ℃ in 5%CO 2Be incubated overnight in the moist incubator.Second day, the plate that will contain cell took out from incubator, culture medium is patted portal, and is dry through on napkin, patting gently then.50 μ L fresh growth medium are accepted in each hole subsequently.(RPMI 1640 measuring culture medium; The 10%FBS complete medium) carries out series dilution in 1: 4 (200nM to 1.9 * 10 of (Q)-hRS7 in -4NM), with triplicate (final concentration 100nM to 0.95 * 10, hole to correspondence -4NM) add in (50 μ L/ hole).At 4 ℃ of following incubations after 1.5 hours; With plate centrifugal 2 minutes with 600 * g; After removing culture medium, it is carried out trace drying (blotted dry) on napkin,, washed in centrifugal 2 minutes with 600 * g then through in each hole, adding the ice-cold culture medium of 150 μ L.Remove culture medium, plate is carried out the trace drying.With 1: 20, the GAH antibody that 000 dilution factor uses HRP-to put together added institute porose (100 μ L/ hole) with it subsequently.For the background contrast, one group of hole is only accepted cell+two and is resisted.With plate 4 ℃ of following incubations 1 hour.Then, plate is centrifugal dry with trace.Use freezing culture medium washed cell 2 times subsequently, wash the 3rd time with freezing PBS then.After washing each time, the process of repeated centrifugation, culture medium removal and plate trace.
Behind last washing step, (KPL, Gaithersburg MD) add institute porose (100 μ L/ hole), use the Envision plate reader to read the luminous of plate with LumiGLO.Use GraphPad Prism software analysis data to measure apparent affinity (apparent affinity), it is the concentration of corresponding half maximum saturation.In each experiment, hRS7 and hLL1 are comprised respectively as positive and isotype contrast.Selectively, use reagent, scheme and the software of manufacturer, (Hayward CA) goes up through flow cytometry and measures combining of (Q)-hRS7 and human carcinoma cell line for Guava Technologies, Inc. at Guava PCA.For each cell line, use hRS7 and hLL1 to carry out similar research coequally.In brief, obtain about 5 * 10 of different cell lines to be analyzed 5Individual cell is suspended in it among PBS/1%BSA (bovine serum albumin).With cell centrifugation, be resuspended among the PBS/1%BSA that 100 μ L contain 10 μ g/mL (Q)-hRS7, hRS7 or hLL1,4 ℃ of following incubations 45 minutes.After with PBS/1%BSA washing 2 times (carrying out centrifugal each time after the washing), cell is resuspended to the GAH that 50 μ L put together FITC, Fc specific antibody (1: 25 dilution factor) is then 4 ℃ of following incubations 30 minutes.Passing through the flow cytometry cell with PBS/1%BSA washing 2 times and after being suspended among the 0.5mL PBS/1%BSA.For dead cell is separated with living cells, add 1 μ g/mL propidium iodide.For analyzing each time, need 10,000 analyses.
Cell proliferating determining with tumor cell inoculation at 96 orifice plates (1 * 10 4Individual cells/well) in, with it with 0.01 to 100nM trial target incubation 72 hours.According to the scheme of manufacturer, use solubility tetrazolium salts MTS [3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxyl methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] to measure the number of living cells subsequently.Use GraphPad Prism software analysis from the data of dose-response curve to obtain EC50 value when suppressing generation (50% concentration).
CFA makes cell experience trypsin treatment, is coated on 60-mm culture dish (1 * 10 then 3Individual cell) in.Handle cell with each trial target, make it form colony.Add the fresh culture that contains trial target per 4 days 1 time, behind the incubation in 2 weeks that colony is fixing in 4% formaldehyde, dye with Jim Sa (Diemsa).The colony that surpasses 50 cells at the microscopically counting.
The internalization research of carrying out through fluorescence microscopy places the ME-180 cell (2000 cells are in 500 μ L/ holes) 8 hole Lab-TekTM II to cultivate chamber microscope slide (NalgeNunc International; Naperville; IL) in, with (Q)-hRS7 (10 μ g/mL) or hRS7 (6 μ g/mL) 37 ℃ of following incubations 16 hours.At room temperature carry out all subsequent steps.After with the PBS/2%BSA washed twice or with the PBS/2%BSA washing, use the 0.1M glycine then 2 times, after pH 2.5 (500 μ L, the 2 minutes) washing 2 times; Cell is fixed 15 minutes in 4% formalin; With PBS washing 2 times, use mouse anti-Rap mAb then, then survey with the GAM that puts together PE, Fc specific antibody; Or directly survey with the GAH that puts together FITC, Fc specific antibody, show the location of (Q)-hRS7 or hRS7 to use fluorescence microscope.
Illustrate the secondary research of the Subcellular Localization of (Q)-hRS7 as follows.To put together the HTrf (hTf) of Alexa Fluor 568 and (Q)-hRS7 (10 μ g/mL) or hRS7 (6 μ g/mL) adds together and places (3000 cells are in 500 μ L/ holes) 8 holes to cultivate the MDA-MB-468 human breast cancer cell of chamber microscope slide.Be 37 ℃ of following incubations after 2 hours, washing and fixed cell were at room temperature handled 15 minutes with the GAH IgG that puts together Alexa Fluor 488 then as stated.After with PBS washing 2 times, at room temperature handled cell 15 minutes with Hoechst 33258, washed cell, inspection cell under fluorescence microscope subsequently.
The BALB/c mouse that toxicity in vivo is accepted to test with (Q)-hRS7 intravenous injection of the various dose of scope from 25 to 400 μ g/ mices for the first time is (female; 7 ages in week, Taconic Farms, Germantown; NY), monitor the toxic visible volume body weight change of seeking peace every day.Maximum tolerated dose (MTD) be defined as do not have dead take place and lose weight be 20% or processing still less before maximum dose level during the weight of animals (about 20g).The animal of painless execution experience poisonous effect.
The therapeutic efficiency of tumor-bearing mice is with 1 * 10 7The female NCr of the about 20g of the Calu-3 people NSCLC cell subcutaneous vaccination athymism nu/nu mice (when receiving from the Taconic farm be 5 week ages) of isozygotying is measured length * wide its tumor growth of monitoring of tumor through measuring the footpath device.Gross tumor volume is calculated as (L * W2)/2.In case tumor reaches the about 0.15cm of size 3, just animal is divided into processed group, every group of 5 animals.Treatment is made up of the injection of the 25 μ g that intravenous injection or two minor ticks of single 50 μ g (Q)-hRS7 were used in 7 days.Matched group is accepted saline.Monitor the toxic sign of animal every day, the genuine painless execution mice of people is if the tumor size reaches above 2.0cm 3Or becoming ulcer, so said animal is considered to yield to PD.In addition, surpass 20% initial body weight or become dying if mice alleviates, so painless execution they.Utilize GraphPad Prism software, use Kaplan-Meier curve (log-rank analysis) to analyze survival data.On P＜0.05, it is statistically evident that difference is considered to.
The result
Purity and molecule integrityShow that through size exclusion HPLC (Q)-hRS7 is made up of the unimodal (not shown) of the observed retention time (7.8 minutes) of the indication molecular size bigger than IgG.(Q)-purity of hRS7 also is confirmed through on reduced form SDS-PAGE, only observing 2 bands (a treaty 50kDa, owing to the heavy chain of hRS7, another treaty 37KDa is owing to the L chain that merges Rap (Q)) (not shown).
Binding analysisThrough the reactivity of ELISA entry evaluation (Q)-hRS7 with the cell line of expressing Trop-2, (both's generation is about 2 times apparent dissociation constant (K of the apparent dissociation constant of hRS7 with Calu-3 (Fig. 2 B) for PC-3 (Fig. 2 A) D) (0.28nM is to 0.14nM)) and this reactivity obtain the proof.Do not observe combination for the negative 22Rv1 of Trop-2-(Fig. 2 C).In the cell line that 10 are expressed Trop-2 altogether, utilize flow cytometry to carry out research subsequently, (not shown) shows that in fact the combination character of (Q)-hRS7 not there are differences with the character that combines of hRS7 as a result.
The RNA enzymatic activityThe IVTT algoscopy is measured the inhibition of the protein synthesis that is caused by the RNA enzymatic degradation because of mRNA.Like what show among Fig. 3 A, (Q)-hRS7 and rRap have comparable RNA enzymatic activity, yet do not observe enzymatic activity for hRS7 in this cell-less measurement method.Through using yeast tRNA as substrate, we estimate rRap with (Q)-kcat/Km (10 of hRS7 9M -1s -1) be respectively 4.10 (± 0.42) and 1.98.Therefore, be about 50% of rRap, this 40% catalytic efficiency similar (people such as Newton, Blood2001 with the LL2-ranpirnase of comparing of report with natural Rap based on the catalytic efficiency of (the Q)-hRS7 of the concentration of Rap; 97:528-35).Initial rate is shown among Fig. 3 B the curve of the concentration (from one group of representative experiment) of tRNA.
Vitro cytotoxicityThe result who measures based on MTS, (Q)-hRS7 resists ME-180 (Fig. 4 A), T-47D (Fig. 4 B), MDA-MB-468 and Calu-3 most strenuously, the EC50 value is respectively 1.5,2.0,3.8 and 8.5nM.Measure through MTS; For being lower than growth inhibiting those cell lines of 50% in the last demonstration of 100nM (Q)-hRS7, we also carry out colony and measure to confirm that (Q)-hRS7 has the cytotoxicity (not shown) to DU-145, PC-3, MCF7, SK-BR-3, BxPC-3, Capan-1 and SK-OV-3 on 10nM or 100nM.The representative result that has shown DU-145 (Fig. 4 C) and PC-3 (Fig. 4 D).In two mensuration, the 100nM that is combined in of hRS7, rRap and hRS7 and rRap has upward shown minimum cytotoxicity (if any) in all cell lines of being assessed.The negative AsPC-1 of Trop-2 has resistance to (Q)-hRS7 in two mensuration.
Internalization and Subcellular LocalizationFor washing with stripping film fixed sample after the bonded protein (not shown), shown the internalization of (Q)-hRS7 to ME-180 cell clearly with PBS/0.2%BSA or with low pH glycine buffer.The distribution pattern of (Q)-hRS7 in ME-180 in the cell; Directly detected or arrive via mouse anti-Rap IgG indirect detection like the GAH through puting together FITC through the GAM that puts together PE; Manifest almost completely identical, this shows (Q)-hRS7 (not shown) that after getting into these cells, still is kept perfectly.Use fluorescently-labeled hTf further to survey (Q)-hRS7 in the intracellular Subcellular Localization of MDA-MB-468 as the labelling and the Hoechst 33258 (its staining cell nuclear) of recycling endosome.According to the result, it is apparent that when at 37 ℃ of following incubations after 2 hours, (Q)-hRS7 occupies the identical subcellular location (not shown) among the MDA-MB-468 with hTf.Like desired (data not shown), except its by PE-GAM/ anti--Rap manifested, hRS7 showed the internalization characteristic similar with (Q)-hRS7 in two kinds of cell lines.
MTD in the miceWe are the MTD that measures (Q)-hRS7 in the normal BALB/c mouse of single intravenous injection of 50 μ g to 100 μ g being given.Other 2L-Rap-X or 2L-Rap (the Q)-X fusion rotein that produce up to now have similar MTD scope.In addition, we are 80 μ g through (Q)-hRS7 of giving 20 μ g (q5d * 4) in 4 times per 5 days and having measured multiple injection MTD in the first SCID mice of accepting experiment.
Therapeutic efficiency in the tumor-bearing miceLike what show among Fig. 5 A; Utilize arbitrary processing (single dose of (Q)-hRS7; The dosage of the 25 μ g that 50 μ g or two intervals gave in 5 days) compare with untreated control and suppressed the growth (P＜0.019) of Calu-3 xenograft with showing, mean survival time increased to 96 days (P＜0.01 from 55 days; Fig. 5 B).
Discuss
With from plant or bacterial origin (Kreitman, AAPS J 2006; Suitable degree (people such as Pastan, Nat Rev Cancer 2006 have been accomplished or proceeded to the immunotoxin of 8:E532-51) toxin preparation (to the clinical trial of the treatment of cancer of said toxin; 6:559-65; People such as Pastan, Annu Rev Med 2007; 58:221-37; Kreitman, BioDrugs 2009; 23:1-13)) compare the favourable aspect of antibody target RNA enzyme (being also referred to as the immune ribonucleic acid enzyme) (people such as De Lorenzo, FEBS Lett 2002; 516:208-12; Cancer Res 2004; Be gentle relatively 64:4870-4), most ofly be used to treat malignant hematologic disease and targeted constituent by exploitation and give (people such as Schirrmann, Exp Opin Biol Ther 2009 by some form of scFv; 9:79-95).Up to now, in having the patient of any cancer, do not assess the immune ribonucleic acid enzyme.
Two difficulties pointing out in the clinical development of other plant or microorganism immunotoxin are toxicity and immunogenicities of not expecting.For the observed common tissue toxicity of these immunotoxins comprise vascular leak syndrome (VLS), hemolytic uremic syndrome (HUS) and liver toxicity (Kreitman, BioDrugs 2009; 23:1-13).Bonded structural motif (x) D (y) through identify being responsible for ricin A-chain or IL-2 and endotheliocyte does not exist in the native sequences of Rap (Q), and hRS7 not with HEC's cross reaction.We thus think that (Q)-hRS7 causes that the probability of VLS is very little.(it has caused possibly pay close attention to (people such as Yokota, Cancer Res1992 to the infiltration not too fast of tumor to large-sized (Q)-hRS7 (about 180kDa); 52:3402-8)) should stop its removing of passing through kidney and mitigation that the risk of HUS takes place.As for liver toxicity; We point out; BL22, by merge the reorganization of forming to the stable Fv of disulfide bond of the RFB4 of PE38 anti--CD22 immunotoxin and similar toxin for example LMB-2 (anti--Tac (Fv)-PE38) also has extremely low MTD because of non-specific liver toxicity in mice; Yet reported BL22 and be (people such as Kreitman, N Engl J Med 2001 safely and effectively in the patient's who suffers from hairy cell leukemia clinical trial; 345:241-7).Therefore, usually in mice observed dose limitation liver toxicity possibly seldom show at philtrum (Kreitman, BioDrugs 2009; 23:1-13).On the other hand, immunogenicity is more common problem.The immunotoxin that most of genetic engineering of in the cancer patient, having assessed is reelected is induced strong humoral immune reaction, and this has shortened serum half-life and has hindered further uses.In laboratory animal, tested several kinds and reduced immunoreactive method, for deoxyspergualin (people such as Pai, Cancer Res 1990; 50:7750-3) and CTLA4Ig (people such as Sieall, J Immunol1997; 159:5168-73) more existing successful reports, and clinical trial (Frankel, the Clin Cancer Res2004 with this type of and other immunosuppressant of immunotoxin combination proposed; 10:13-5).(Q)-and the immunogenicity of hRS7 is lower, as if because it comprises the fusions of humanized antibody and toxin, said fusions is induced antibody response (people such as Mikulski, J Clin Oncol 2002 hardly in the patient; 20:274-81).
The cytotoxicity of immunotoxin needs it to get into target cell, migrates to cytosol subsequently.Though reported ranpirnase (people such as Rodriguez, J Cell Sci 2007; 120:1405-11; Haigis and Raines, J Cell Sci 2003; 116:313-24; People such as Wu, J Biol Chem1995; 270:17476-81) with other RNA enzyme (people such as Wu, J Biol Chem1995; 270:17476-81; People such as Leich, J Biol Chem 2007; 282:27640-6; People such as Bracale, Biochem J 2002; 362:553-60) and comprise merge the pure man anti--ErbB2scFv (people such as De Lorenzo, Cancer Res 2004; 64:4870-4; FEBS Lett2007; 581:296-300) or the people anti--CD30 scFv-Fc (people such as Menzel, Blood2008; Approach in the cell after the internalization of the immune ribonucleic acid enzyme of human pancreas RNA enzyme 111:3830-7) does not occur but understand yet fully.The experiment of our internalization shows when being added to MDA-MB-468 after 2 hours when checking; (Q)-hRS7 and hTf locate altogether; This shows that (Q)-hRS7 can directly come out to get into cytosol from endosome, like (Haigis and Raines, the J Cell Sci 2003 for ranpirnase proposed; 116:313-24).Between anti--Rap and the Anti-Human Fc f in ME-180 the close similarity for the viewed fluoroscopic image of (Q)-hRS7 in the cell further show (Q)-hRS7 ability that protease inhibitor is degraded in the endocytosis process.
Although when MTS measured measurement through 3 days; The vitro efficacy of finding (Q)-hRS7 can change (this can part owing to the difference cell in Path selection) between the cell line of expressing Trop-2; But, show that clearly there is the cytotoxicity to all cells system in (Q)-hRS7 on 10nM through using CFA.Except the cytotoxicity of its strong external anti-multiple cancerous cell line; Show that also (Q)-hRS7 is effective in the growth of the Calu-3 people's pulmonary carcinoma xenograft that suppresses nude mouse; Thereby verified anti-tumor in vivo activity and the stability of (Q)-hRS7 and confirmed the fitness (Kreitman, the AAPS J 2006 that are added Trop-2 by the antigen on the solid carcinoma of immunotoxin targeting to present tabulation; 8:E532-51; People such as Pastan, Nat Rev Cancer 2006; People such as Pastan, Annu Rev Med 2007; 58:221-37; People such as Schirrmann, Exp Opin Biol Ther 2009; 9:79-95).
In a word, we proved with humanization anti--Amphibian RNA enzyme that the reorganization of Trop-2 antibody is merged all shows in vitro and in vivo and resists multiple epitheliomatous selectivity and strong cytotoxicity.
Expression and the evaluation of embodiment 2.2L-rap-hLL1-γ 4P
As hereinafter employed, rap representes ranpirnase.
The structure of dHL-IgG4P variant:
The B13-24 cell that comprises the IgG4 gene is available from ATCC (ATCC numbers CRL-11397) and isolation of genomic DNA.Cell washs with PBS, is suspended in digestion buffer (100mM NaCl, 10mM Tris-HCl pH 8.0,25mM EDTA, 0.5%SDS, 0.1mg/ml E.C. 3.4.21.64), 50 ℃ of following incubations 18 hours.With isopyknic phenol/chloroform/isoamyl alcohol extracting sample, use 7.5M NH then 4Ac/100%EtOH precipitates.Through centrifugal recovery genomic DNA, it is dissolved in the TE buffer.Use genomic DNA as template, through pcr amplification IgG4 gene.
Amplification PCR products is cloned into TOPO-TA sequencing vector (Invitrogen), confirms through dna sequencing.The SacII-EagI fragment that contains the CH of IgG1 among the pdHL-hLL2 replaces with the SacII-EagI of TOPO-TA-IgG4 plasmid, thereby produces pdHL2-hLL2-IgG4 (pdHL2-hLL2-γ 4) carrier.
IgG 4The sudden change of-proline
The hinge region of the Ser228Pro sudden change being introduced IgG4 is to avoid the formation of half point.The hinge region 56bp fragment (PstI-StuI) of synthetic sudden change makes its annealing, replaces with IgG then 4The PstI-StuI fragment.This construct causes producing final carrier pdHL2-hLL2-γ 4P.
The structure of pdHL2-hLL1-γ 4P
The Xba-HindIII fragment that the XbaI-HindIII fragment of pdHL2-hLL2-γ 4P is replaced with the pdHL2-hLL1 that contains Vk and VH district is to produce hLL1-γ 4P construct.
The structure of pdHL2-2L-rap-hLL1-γ 4P
4 monomeric flexible connection bodies of glycine-1 serine that use comprises 3 copies are connected to the C-terminal of Rap the N-terminal of the Vk of hLL1.The N-terminal of each bar light chain connects a rap molecule.Carry out the structure of the DNA of this molecule through PCR.The Xba-BamHI fragment of pdHL2-hLL1-γ 4P is replaced with the Xba-BamHI of pBS-2L-rap-hLL1, and (fragment of Xba-targeting sequencing-rap-connector-Vk-BamHI) is to accomplish final carrier pdHL2-2L-rap-hLL1-γ 4P.
Transfection
Carrier DNA (30 μ g) carries out linearisation with the SalI enzyme, then through electroporation (450V) transfection NSO (4 * 10 6Individual cell/mL) or Sp2/0-Ag14 (5 * 10 6The myeloma cell of individual cell/mL).Said cell culture is being supplemented with low-IgG FBS (10%), penicillin (in complete hybridoma-SFM culture medium of 100 units/mL), streptomycin (100 μ g/mL), L-glutaminate (2mM), Sodium Pyruvate (1mM), essential amino acids (100 μ M) and methotrexate (0.1 μ M).Through the ELISA screening positive clone.In brief, dull and stereotyped with the anti--rap antibody sandwich in the PBS culture medium of the 5ug/mL of 50 μ l, under 4 ℃, be incubated overnight.With the PBS washing with after, add cell culture supernatant with the 2%BSA sealing.Goat anti-human IgG .sub.4 the antibody that to put together HRP be used to detect and with OPD with the substrate that acts on colour developing.Read plate at 490nm.The expansion positive colony uses its freezing being used in the future.Clone C6 is accredited as best Producer and is used for further exploitation.
Express and purification
With cell culture in 2 rolling bottles that the 500ml culture medium is housed separately to final cultivation (viablity of 10-20%), through the centrifugal cell of removing.With supernatant liquid filtering, be used for then with the equilibrated protein A pillar of 20mM Tris-HCl/100mM NaCl buffer (pH 8.5).After the loading,, use 100mM sodium citrate buffer solution (pH 3.5) to carry out eluting then to obtain fusion rotein with 100-mM sodium citrate buffer solution (pH 7.0) washing pillar.Use 3MTris-HCl, the peak that pH 8.5 will comprise product is adjusted to pH 7.0, dialyses to 10mM PBS solution.After concentrating, product is filtered through 0.22 μ m filter, store down in 2-8 ℃.Behind the purification, reclaim 16mg from the 1-L culture.
The evaluation of 2L-rap-hLL1-γ 4P
HPLC: proteinic purity of inspection and concentration on HPLC.Observe the single sharp peak of retention time indication molecule 7.7 minutes (not shown)s greater than IgG.
SDS-PAGE: use the 4-20%Tris-glycine gels under reductive condition, to carry out SDS-PAGE.Observe and expect two bands (both are greater than the light chain (about 25kD) of hLL1) (not shown) of the relevant band of heavy chain and molecular weight about 37 and 39kD of the about 50kD of size.Shown the glycosylation (vide infra) of the existence of two light chains owing to rap on the fusion rotein.
Mass spectrography: at Scripps Research Institute, CA carries out mass spectrography through the MALDI-TOF method.Two samples are presented analyzing, and one exists (1.6mg/mL is in 10mM PBS) with native state, and there be (1.6mg/mL is in 1mM HEPES/10mM DTT, in pH 7.5 buffer) in another with reducing condition.Natural sample shows the main peak of a quality 177150, the just in time identical (not shown) of this MW with 1 IgG+2 rap.Reductive sample is gone up at 50560 (corresponding to heavy chains), 38526 and 36700 (corresponding to 2 light chains that contain rap) and is shown 3 main peak (not shown)s.
Western blotting: be present in the purified proteins matter in order to confirm rap, carry out western blotting.Will be under reductive condition from the sample electrotransfer of SDS-PAGE gel to pvdf membrane.After with the 5%BSA sealing, with 1: 10,000 dilution factor or 10ng/ml added mouse anti-rap antibody, incubation 1 hour.After washing, add goat anti-mice Fc antibody of puting together HRP, incubation 1 hour.After washing 6 times, add LumiGloTM (Kirkegaard & Perry Laboratories) substrate, make the Kodak film development.On film, detect corresponding to two bands that merge light chain, thereby confirm that rap is present in (not shown) on two light chains.
Utilize the processing of N-glycosidase: because rap has potential N glycosylation site Asn-X-Thr/Ser, Asn69-Val70-Thr71, therefore the observation of the light chain of two molecular masses with 2kD difference possibly be the inhomogeneous glycosylated result of rap.In order to study this probability, with rap-hLL1 antibody with N-glycosidase (New England Biolabs) incubation under according to the degeneration condition of the recommendation of provider.After the N-glycosidase is handled, be merged into one (moving band faster) corresponding to two bands of two light chains, thereby confirmed that sugared uneven distribution is on SDS-PAGE, to observe the reason of two band (not shown)s.Other proof provides through when Rap (N69Q) (having removed the variant of the Rap of glycosylation site) substitutes the Rap in the recombinant precursor, only observing a Rap fusion light chain (data not shown).
The activity of rap: Bright-Glo is used in the recommendation according to provider TMLuciferase Reporter Assay system (Promega) utilizes
Figure BDA0000139409280000641
Quick Coupled Transcription/Translation system (Promega) test rna enzymatic activity.The principle of this mensuration is to use the inhibition (mRNA degraded) of luciferase reporting systematic survey as the result's of RNA enzymatic activity protein synthesis.With different dilution factors: the chemically conjugated thing (being expressed as K1-LL2-One and PKII-LL2-One) of free rap (0.001-2.5nM), hLL1-rap (0.01-20nM) or hLL2-rap (0.01-20nM) prepares sample.Each sample (5uL) is mixed with 20 μ lTNT master mixture, under 30 ℃ in 96 orifice plates incubation 2 hours, take out 1 μ l from said plate and be used to utilize 50 μ l Bright-Glo TMThe analysis that substrate carries out.Use Excel or Prism Pad software, the result is shown among Fig. 6.Chemically conjugated thing EC for rap-hLL1 and hLL2-One 50Value is for about 300pM, for free rap, is worth and is 30pM.
The competition of WP combinesWP is anti--idiotype antibody of hLL1.Assess the affinity that rap-hLL1 antibody is compared with hLL1 antibody through competitive binding assay to WP.In brief, encapsulate 96 orifice plates, under 4 ℃, be incubated overnight with the WP of 50 μ l 5ug/mL.With 2 different * dilution factor (the final concentration scope is between 0.49 to 1000nM) preparation 3 type of protein sample hLL1, rap-hLL1 or hA20, with said sample with isopyknic 2 * put together mLL1 antibody (whole dilution factor is 1/20, the 000) mixing of HRP.The blended protein example of mLL1 that 50 μ L and above-mentioned are puted together HRP adds in each hole and incubation 1 hour.After washing, add the H that contains the OPD substrate 2O 2, read flat board at the 490nm place.Like what show among Fig. 7, use Excel or Prism Pad mapping software that protein concentration is mapped to absorbance.HA20 (humanization anti-CD 20 antibodies) is used as negative control.According to Fig. 7, clearly rap-hLL1 has the binding affinity similar with hLL1 and negative control hA20 does not have affinity.Use the Raji cell to obtain similar result as the antigen source.
Vitro cytotoxicityIn B cell lymphoma cell line (Daudi) and multiple myeloma cells system (MC/CAR), measure vitro cytotoxicity.Cell (among the 0.1ml 10,000) is placed each hole of 96 orifice plates.After 24 hours, the hLL1 that will dissociate, free rap or rap-hLL1 (10 μ l) add in the suitable hole, with cell under 37 ℃ in incubator incubation 3 days.Use MTS tetrazolium dye determination by reduction method or BrDU colorimetric method to measure cell proliferation.The result is expressed as EC 50, said EC 50Through using the diagram of Prism Pad software to obtain.It is apparent that according to Fig. 8-9 rap-hLL1 is very sensitive to B cell lymphoma cell line (Daudi) and multiple myeloma cells system (MC/CAR).Rap-hLL1 is to Daudi cell and significantly more effective force (cytotoxicity) that the MC/CAR cell is compared, as passes through EC 50Value (Fig. 8 and Fig. 9) reflection.For MC/CAR cell line, on the concentration of test, do not reach EC 50Value.On maximum concentration (56nM), the cell viablity is 57%.HLL1 or free rap showed cell toxicity not in arbitrary cell line itself.
Pharmacokinetics and bio distribution methodLike people such as Sharkey (Int J Cancer.1990; 46:79-85) described; Use 2-(the different sulfur cyanobenzyl of 4-) DTPA (Macrocyclics; Dallas Tex.) is conjugated to diethylene triamine pentacetic acid (DTPA) (DTPA) to obtain DTPA-hLL 1 or DTPA-2L-Rap-hLL1-γ 4P with hLL1 or 2L-Rap-hLL1-γ 4P, uses chlorination respectively 88Y (Los Alamos National Laboratory (Los Alamos, N.Mex.) or chlorination 111(Mass.) labelling DTPA-hLL1 or DTPA-2L-Rap-hLL1-γ 4P are to carry out pharmacokinetics and biodistribution research for Perkin Elmer Life Sciences, Boston.Use 0.001mCi 88Y-DTPA-hLL1 and 0.02mCi 111The female SCID mice that the mixture of In-DTPA-2L-Rap-hLL1-γ 4P (being supplemented with the DTPA conjugate of unlabelled hLL1 or 2L-Rap-hLL1-γ 4P) intravenous injection is accepted to test for the first time (8 ages in week, 18-22g) hLL1 and the 2L-Rap-hLL1-γ 4P of 10 μ g so that every animals received accumulated dose done for oneself.On the time of selecting (1,2,4,16,48,72,168 hour), anesthetized mice group (every group of 5 mices) utilizes cardiac puncture to extract blood sample after the administration.Take out main tissue, weigh, be placed in the container.Counting blood sample and tissue in calibrated gamma counter 111In (passage 120-480) and 88Y (passage 600-2000).Produce the exchange curve to proofread and correct 88Y can be extremely 111Backscatter in the In number window.
Toxicity in vivoAccept the SCID or the BALB/c mouse of testing for the first time with the 2L-Rap-hLL1-γ 4P intravenous injection of the various dose of scope from 25 to 400 μ g/ mices, monitor toxic visible volume every day and seek peace and lose weight.Maximum tolerated dose (MTD) is defined as does not have dead the generation and the maximum dose level into less than the weight of animals before 20% the processing (about 20g) time of losing weight.Put to death and gather in the crops the animal of experience poisonous effect, with its experience histopathological analysis.In the first SCID mice of accepting to test; 100, the single intravenous dosages of the 2L-Rap-hLL1-γ 4P of 150,200,250,300 or 400 μ g causes seriously losing weight of animal and death, but the (not shown) of surviving behind the dosage of all mice experience 25 or 50 μ g.In BALB/c mouse, all mices survive behind the 2L-Rap-hLL1-γ 4P of the single intravenous dosages of experience 30 or 50 μ g but non-100 or 200 μ g (not shown)s.In another experiment, find that the 2L-Rap-hLL1-γ 4P of 75 μ g dosage is deleterious (data not shown) to the SCID mice.Therefore, the MTD of the 2L-Rap-hLL1-γ 4P that gives as bolus injection is 50 to 75 μ g in the SCID mice, is 50 to 100 μ g in BALB/c mouse.Macropathology inspection dead or sacrificed mice shows serious liver and spleen toxicity.Liver is pale asphyxia and spleen shrinkage, less than common size.Histopathological examination shows liver and SN.Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and STB that the blood serum sample of representative mice has the level of rising are illustrated in and have significant liver toxicity on these high doses.
Data analysisFor the vitro cytotoxicity analysis, from the meansigma methods generation dose-response curve of triplicate mensuration, (Advanced Graphics Software, Encinitas Calif.) obtain 50% inhibition concentration (IC to use GraphPad Prism software 50) value.Use non-compartment model analysis programme (noncompartmental analysis program) WinNonlin, 4.1 editions (canonical algorithm Calif.) is analyzed pharmacokinetic data for Pharsight, Mountain View.Said program utilizes linear trapezoid method then, uses area (AUC) under the linear interpolation calculated curve.Suppose first order kinetics, according to t1/2 (t 1/2 β) calculating elimination rate constant (k β).Utilize GraphPad Prism software, use Kaplan-Meier curve (log-rank analysis) to analyze survival research.Difference o'clock is statistically evident in P＜0.05.
Pharmacokinetics and bio distribution dataIn the first SCID mice of accepting to test, measure pharmacokinetics and the bio distribution of radiolabeled hLL1 and 2L-Rap-hLL1-γ 4P.Put together hLL1 and 2L-Rap-hLL1-γ 4P with DTPA, and use respectively 88Y with 111In carries out the labelling spike.Like what show among Figure 10, 111The 2L-Rap-hLL1-γ 4P of In-labelling show with 88The blood two-phase property clearance rate that the hLL1 of Y-labelling is similar is characterized in that initially distributing fast (α) and slower subsequently removing (β) mutually again.Compare with hLL1 (4 hours), observe the slower a little half-life for 2L-Rap-hLL1-γ 4P (5.1 hours).Use the data point that surpasses 5 hours to calculate t 1/2 β, k β, AUC, average retention time (MRT), apparent volume of distribution (V d) and clearance rate (Cl), and the value of these parameters is shown in Table 2. 111The tissue absorption of the 2L-Rap-hLL1-γ 4P of In-labelling with 88The tissue absorption of the hLL1 of Y-labelling similar (data not shown).
2L-Rap-hLL1-γ 4P that the radiolabeled DTPA-conjugate of table 2. use is measured in the SCID mice and the pharmacokinetic parameter of hLL1
Parameter Unit 88Y-DTPA-hLL1 111In-DTPA-2L-Rap-hLL1-γ4P
T 1/2，β h 103 113
k β 1/h 0.0067 0.0061
Cl mL/h 0.025 0.024
Vd mL 3.8 3.9
MRT h 140 156
AUC h*μg/mL 393 418
Therapeutic efficiency in the tumor-bearing mice
Therapeutic efficiency in the tumor-bearing mice: with 1.5 * 10 7The female SCID mice of individual Daudi cell intravenous injection (in 8 ages in week, 18-22g), 8 to 9 every group, is accepted processing after one day.Check the hind leg paralysis of mice every day, and weigh once weekly.Before hind leg paralysis takes place or loses 20% their processing in animal during weight, painless execution they.Stop each group treatment experiment after 180 days.
Like what show among Figure 11, untreated mice (PBS group) is all dead in 30 days, and mean survival time (MST) is 28 days.The MST of matched group that accepts the mixture (representing the compositions of the constitutive protein matter among the 50 μ g2L-Rap-hLL1-γ 4P) of hLL1-γ 4P (43.2 μ g) and Rap (6.6 μ g) is 70 days (P＜0.0001 is with respect to the PBS group).On the contrary, all mices of accepting the single injection of 5 or 15 μ g2L-Rap-hLL1-γ 4P (MST＞180 day of all surviving and surpass 100 days; P=0.0005 is with respect to the group of component processing), and have only a mice when stopping, losing in every group near research.When after 180 days, stopping research, the mice of the single injection of 90 % acceptance 5,15,30,40 or 50 μ g 2L-Rap-hLL1-γ 4P obtains curing.MST that it should be noted that the mice of the single injection of accepting 1 μ g is 92 days, with untreated group 28 days compare (P＜0.0001), and the increase of expression 230%.
The DNA's of the Codocyte toxicity RNA enzyme of PCR-amplification is synthetic
Utilize automatization's dna synthesizer to synthesize 139-aggressiveness DNA nucleotide, ONCO-N, it has the sense strand sequence of the N terminal sequence (46 aminoacid) of coding reconstitution cell toxicity RNA enzyme:
5 '-TGG CTA ACG TTT CAG AAG AAA CAT ATC ACG AAT ACACGA GAT GTA GAC TGG GAC AAT ATA ATG TCT ACG AAT CTGTTT CAC TGT AAG GAT AAG AAT ACC TTT ATA TAC AGT CGGCCA GAG CCT GTA AAG GCT ATC TGT A-3 ' (SEQ ID NO:88); As template, utilize following primer to carry out pcr amplification it.
ONNBACK
5′-AAG?CTT?CAT?ATG?CAG?GAT?TGG?CTA?ACG?TTT?CAG?AAGAAA-3′(SEQ?ID?NO：89)
ONNFOR
5′-CTT?ACT?CGC?GAT?AAT?GCC?TTT?ACA?GAT?AGC?CTT?TACAGG?CTC?TG-3′(SEQ?ID?NO：90)
The double-stranded PCR product of gained comprises the cDNA sequence of N end half 54 amino acid residues partly of Codocyte toxicity RNA enzyme.ONNBACK comprises restriction enzyme site HindIII and NdeI and is connected (NdeI site) sub-clone in the frame and goes into bacterial expression vector to help sub-clone and go into staging vector (staging vector) or to make.The NruI site is integrated into the ONNFOR primer to help holding the cDNA of half part to be connected with the C of Codocyte toxicity RNA enzyme in the frame.
Similarly, synthetic 137-aggressiveness DNA nucleotide, ONCO-C, it has the sense strand sequence of the C terminal sequence (46 aminoacid) of Codocyte toxicity RNA enzyme:
5 '-TGC TGA CTA CTT CCG AGT TCT ATC TGT CCG ATT GCAATG TGA CTT CAC GGC CCT GCA AAT ATA AGC TGA AGA AAAGCA CTA ACA AAT TTT GCG TAA CTT GCG AGA ACC AGG CTCCTG TAC ATT TCG TTG GAG TCG GG-3 ' (SEQ ID NO:91), utilize the following primer PCR said sequence that increases.
ONCBACK
5′-ATT?ATC?GCG?AGT?AAG?AAC?GTG?CTG?ACT?ACT?TCCGAG?TTC?TAT-3′(SEQ?ID?NO：92)
ONCFOR
5′-TTA?GGA?TCC?TTA?GCA?GCT?CCC?GAC?TCC?AAC?GAA?ATGTAC-3′(SEQ?ID?NO：93)
Final double-stranded PCR product comprises 51 amino acid whose cDNA sequences of remaining C end half part of Codocyte toxicity RNA enzyme.The NruI site allow in the frame be incorporated into ONCBACK in the N end half of DNA of pcr amplification partly be connected.Being used for sub-clone goes into the termination codon and the BamHI restriction enzyme site of staging vector or expression vector and is included in the ONCFOR sequence.
After handling with suitable restricted enzyme, the N end of Codocyte toxicity RNA enzyme and the DNA of C end half pcr amplification partly are connected in the NruI site, sub-clone is gone into staging vector then, for example from the pBluescript of Stratagene.The sequential coding that connects has 105 amino acid whose polypeptide of N-terminal Met.
The humanization of the clone of LL2 and MN-14V-region sequence and LL2 and MN-14
The V region sequence of hLL2 and hMN-14 is announced.People such as Leung, Mol.Immunol., 32:1413 (1995); The 5th, 874, No. 540 United States Patent (USP)s.Use the method for announcement and VK and the VH sequence of primer PCR amplification LL2 and MN-14.The sequence analysis of the DNA of pcr amplification is shown their encode protein of common antibody VK and VH domain.The LL2 of PCR-based amplification and the chimeric antibody of MN-14 sequence construct show the immunoreactivity that can compare with their parental generation antibody, thereby have confirmed the verity of the sequence of acquisition
The sequence analysis of LL2 antibody is presented at the glycosylation site of the N-connection that has the VK suspension in framework-1 zone.Mutation research shows that the glycosylation on the VK-suspension site is not to keep the necessary (not shown) of the immunoreactivity of antibody.Under the situation that does not comprise the FR-1 glycosylation site, the REI frame sequence is as the support of transplanting light chain CDR, and EU/NEWM is used to transplant the heavy chain CDR of LL2.The immunoreactivity (not shown) of humanization LL2 (hLL2) is through the suitable (not shown) of immunoreactivity of demonstration and Mus and chimeric LL2.The internalization speed of LL2 does not receive chimericization of antibody or humanization to influence (not shown).
The structure of the gene of the fusion rotein of coding humanization LL2 and cytotoxicity RNA enzyme
VH and the VK sequence of hLL2 are come the gene through standard pcr assembling hLL2-scFv as template.The N that-1 locational Met start codon is incorporated into the VL gene holds, and is connected to the VH domain through 16 amino acid whose connectors.The carboxyl terminal that the afterbody of 6 histidyl-residues is included in the VH chain is to help through the metal chelate chromatography purified fusion protein.
Make up the immunotoxin fusion protein gene of ranpirnase-hLL2scFv in a similar manner through restricted degraded and method of attachment.The cDNA sequence, when expressing, coding has the fusion rotein that is connected to the terminal ranpirnase of LL2 VL sequence of N through short connector.Have multiple connector, it can be inserted between cytotoxicity RNA enzyme C-terminal and the VL domain N end.Preferred connector is the aminoacid sequence TRHRQPRGW (SEQ ID NO:94) from the C end position 273-281 of PE (PE).Shown that this sequence is PE and is slit into the recognition site of active fragment by subtilisin cell inscribe, cutting occurs between the G and W residue of sequence.People such as Chiron, J.Biol.Chem., 269:18167 (1994).Being integrated with of this sequence helps the release of competent cell toxicity RNA enzyme after the fusion immunotoxin internalization.Selectively, be used for 13 amino acid residue spacers that the amino acid residue 48-60 by the fragment B of staphylococcal protein A of the structure of EDN-scFv fusions forms and alternately be used to allow flexibly connecting between cytotoxicity RNA enzyme and the scFv.People such as Tai, Biochemistry, people such as 29:8024 (1990) and Rybak, Tumor Targeting, 1:141 (1995).
The structure of the gene of the fusion rotein of coding humanization MN-14 and ranpirnase
MN-14 scFv produces through the cDNA of pcr amplification from humanization MN-14 transfectoma.The connector that is used for MN-14 scFv is 15 amino acid whose connectors and is oriented to VL-connector-VH.After confirming DNA sequence, strand construct sub-clone is gone into the eukaryote expression vector, and transfection is to the suitable mammalian host cell that is used for expressing.
Also prepare another strand construct.This utilizes the heavy chain and the light chain of opposite 5 '-3 ' orientation to prepare, with its be assemblied in pCANTABE5E (Pharmacia Biotech, Piscataway, N.J.) in, in phage, express then.The specificity of expressing the recombinant phage of this scFv combines to prove (not shown) through ELISA.
The scFv sequence is used to make up ranpirnase-MN-14 fusion rotein, and it is terminal that ranpirnase is connected to the VL sequence of N through connector.Obtain the dna fragmentation of coding ranpirnase as stated.Between ranpirnase sequence and scFv, use 23 amino acid whose connectors.People such as Kurucz (1995).
Stop and lock
Embodiment 3. is used for the general strategy of generation module Fab subunit
The Fab module can be prepared into the fusion rotein that comprises DDD or AD sequence.For each Fab fusion rotein is developed independently transgenic cell line.When preparation, if desired, module can be carried out purification or is kept in the supernatant of cell culture.After the preparation, any DDD 2But combination in any between module and any AD module is to produce the DNL construct.
Plasmid vector pdHL2 has been used to produce multiple antibody and based on the construct of antibody.Referring to people such as Gillies, J Immunol Methods (1989), 125:191-202; People such as Losman, Cancer (Phila) (1997), 80:2660-6.Synthesizing 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 all is identical, and (VH and VL) sequence there are differences only in the variable region.Use biology tool well known by persons skilled in the art, can these IgG expression vectors be converted into Fab-DDD or Fab-AD expression vector.In order to produce the Fab-DDD expression vector, the coded sequence of the hinge region of heavy chain, CH2 and CH3 domain replaces with the sequence (being called DDD1) of preceding 44 residues of Gly-Ser connector and the people RII α of preceding 4 residues of the said hinge of coding, 14 residues.In order to produce the Fab-AD expression vector; The sequence of the hinge region of IgG, CH2 and CH3 domain replaces with the sequence (being called AD1) of synthetic AD of 17 residues of Gly-Ser connector and AKAP-IS by name of preceding 4 residues, 15 residues of the said hinge of coding; This sequence system uses bioinformatics and peptide array technique to produce, and demonstration very high with the bonded affinity of RII α dimer (0.4nM).Referring to people Proc.Natl.Acad.Sci. such as Alto, U.S.A (2003), 100:4445-50.
Designed two shuttle vectors, be beneficial to the IgG-pdHL2 carrier is converted into Fab-DDD1 or Fab-AD1 expression vector, be described below.
The preparation of CH1
CH1 domain system uses the pdHL2 plasmid vector as template, obtains through pcr amplification.Left side PCR primer is made up of the upper reaches (5 ') of CH1 domain and SacII restriction endonuclease site (be CH1 coded sequence 5 ').The right side primer is made up of the sequence of preceding 4 residues of coding hinge and short connector (latter two codon comprise Bam HI restriction enzyme site).
5 ' of CH1 left side primer
5’GAACCTCGCGGACAGTTAAG-3’(SEQ?ID?NO：63)
CH1+G 4 The S-Bam right side
5’GGATCCTCCGCCGCCGCAGCTCTTAGGTTTCTTGTCCACCTTGGTGTTGCTGG-3’(SEQ?ID?NO：64)
410bp pcr amplification primer is cloned into pGemT PCR cloning vehicle, and (Promega, Inc.), screening and cloning is to obtain the insert of T7 (5 ') direction then.
(G 4S) 2The structure of DDD1
Use Sigma Genosys (Haverhill, UK) synthetic duplex oligonucleotide (called after (G 4S) 2DDD1), with the aminoacid sequence of encoding D DD1, there are 11 residues of connection peptides said DDD1 front, and wherein preceding two codons comprise the BamHI restriction enzyme site.Termination codon gives with the EagI restriction enzyme site and is positioned at 3 ' end.The encoded polypeptides sequence is as follows.
GSGGGGSGGGGSHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：65)
Synthesize two oligonucleotide (on the called after RIIA1-44 and under the RIIA1-44, their 3 ' end has the overlapping of 30 base pairs) (Sigma Genosys), and they are combined to comprise 154 base pairs placed in the middle of 174bp DDD1 sequence.Oligonucleotide is annealed, use the Taq polymerase to carry out primer extension reaction then.
RIIA1-44 is last
5’GTGGCGGGTCTGGCGGAGGTGGCAGCCACATCCAGATCCCGCCGGGGCTCACGGAGCTGCTGCAGGGCTACACGGTGGAGGTGCTGCGACAG-3’(SEQ?ID?NO：66)
Under the RIIA1-44
5’GCGCGAGCTTCTCTCAGGCGGGTGAAGTACTCCACTGCGAATTCGACGAGGTCAGGCGGCTGCTGTCGCAGCACCTCCACCGTGTAGCCCTG-3’(SEQ?ID?NO：67)
After the primer extension, use following primer that said duplex is increased:
A G4S Bam-left side
5’-GGATCCGGAGGTGGCGGGTCTGGCGGAGGT-3’(SEQ?IDNO：68)
It is right that 1-44 stops Eag
5’-CGGCCGTCAAGCGCGAGCTTCTCTCAGGCG-3’(SEQ?IDNO：69)
This amplimer is cloned into pGemT, screens the insert of T7 (5 ') direction then.
(G 4S) 2The structure of-AD1
Synthetic duplex oligonucleotide (called after (G 4S) 2-AD1) (Sigma Genosys), with the aminoacid sequence of coding AD1, there are 11 residues of connection peptides said AD1 front, and wherein preceding two codons comprise the BamHI restriction enzyme site.Termination codon and EagI restriction enzyme site are positioned at 3 ' end.The encoded polypeptides sequence is as follows.
GSGGGGSGGGGSQIEYLAKQIVDNAIQQA(SEQ?ID?NO：70)
Synthetic two complementations also have eclipsed oligonucleotide (on the called after AKAP-IS and under the AKAP-IS).
AKAP-IS is last
5’GGATCCGGAGGTGGCGGGTCTGGCGGAGGTGGCAGCCAGATCGAGTACCTGGCCAAGCAGATCGTGGACAACGCCATCCAGCAGGCCTGACGGCCG-3’(SEQ?ID?NO：71)
Under the AKAP-IS
5’CGGCCGTCAGGCCTGCTGGATGGCGTTGTCCACGATCTGCTTGGCCAGGTACTCGATCTGGCTGCCACCTCCGCCAGACCCGCCACCTCCGGATCC-3’(SEQ?ID?NO：72)
Use following primer that said duplex is carried out pcr amplification:
A G4S Bam-left side
5’-GGATCCGGAGGTGGCGGGTCTGGCGGAGGT-3’(SEQ?IDNO：73)
It is right that AKAP-IS stops Eag
5’-CGGCCGTCAGGCCTGCTGGATG-3’(SEQ?ID?NO：74)
This amplimer is cloned into the pGemT carrier, screens the insert of T7 (5 ') direction then.
Connect DDD1 and CH1
Use BamHI and NotI restricted enzyme that the 190bp fragment of encoding D DD1 sequence is downcut from pGemT, then this fragment is connected to the same loci of CH1-pGemT, to produce shuttle vector CH1-DDD1-pGemT.
Connect AD1 and CH1
The 110bp fragment of using BamHI and NotI will contain the AD1 sequence is downcut from pGemT, then this fragment is connected to the same loci of CH1-pGemT, to produce shuttle vector CH1-AD1-pGemT.
CH1-DDD1 or CH1-AD1 are cloned into the carrier based on pdHL2
Use this modularized design, CH1-DDD1 or CH1-AD1 all can be contained in any IgG construct in the pdHL2 carrier.Through removing the SacII/EagI restriction fragment (CH1-CH3) of pdHL2, and it is replaced with the CH1-DDD1 that downcuts from corresponding pGemT shuttle vector or the SacII/EagI fragment of CH1-AD1, whole CH promptly replaces with one of above-mentioned construct.
The terminal DDD domain of N-
The position of DDD or AD is not limited to the carboxyl terminal of CH1.Transformed the aminoterminal construct that DDD1 sequence wherein is connected to the VH domain.
Embodiment 4.DNL expression vector
The structure of h679-Fd-AD1-pdHL2
H679-Fd-AD1-pdHL2 is the expression vector that is used to produce h679 Fab, and wherein AD1 is coupled to the carboxyl terminal of the CH1 domain of Fd through the flexible Gly/Ser peptide spacer of being made up of 14 amino acid residues.The SacII/EagI fragment based on the carrier of pdHL2 that will comprise the variable region of h679 replaces with CH1-AD1 fragment (being downcut from the CH1-AD1-SV3 shuttle vector by SacII and EagI), and this carrier promptly 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 that is used to produce fusion rotein C-DDD1-Fab-hMN-14, and in said fusion rotein, DDD1 is connected to the CH1 carboxyl terminal of hMN-14 Fab through flexible peptide spacer.Plasmid vector hMN14 (I)-pdHL2 (being used to produce hMN-14 IgG) is through digesting to remove the CH1-CH3 domain with SacII and EagI restriction endonuclease; Insert CH1-DDD1 fragment (downcutting from the CH1-DDD1-SV3 shuttle vector) then, promptly be converted into C-DDD1-Fd-hMN-14-pdHL2 by SacII and EagI.
The structure of N-DDD1-Fd-hMN-14-pdHL2
N-DDD1-Fd-hMN-14-pdHL2 is used to produce the dimeric expression vector of stablizing that comprises two copy fusion albumen N-DDD1-Fab-hMN-14; In said fusion rotein, DDD1 is connected to the amino terminal of the VH of hMN-14 Fab through flexible peptide spacer.As follows said expression vector is transformed.Two primers shown in below using carry out pcr amplification to the DDD1 domain.
A DDD1 Nco left side
5’CCATGGGCAGCCACATCCAGATCCCGCC-3’(SEQ?IDNO：75)
DDD1-G 4 S Bam is right
5’GGATCCGCCACCTCCAGATCCTCCGCCGCCAGCGCGAGCTTCTCTCAGGCGGGTG-3’(SEQ?ID?NO：76)
As the result of PCR, the coded sequence that comprises the part of BamHI restriction enzyme site in NcoI restriction enzyme site and the connector promptly is connected to 5 ' and 3 ' end respectively.170bp pcr amplification primer is cloned into the pGemT carrier, and screening and cloning is to obtain the insert of T7 (5 ') direction then.The 194bp insert is downcut from said pGemT carrier by NcoI and SalI restricted enzyme, and is cloned into said SV3 shuttle vector (using identical enzymic digestion to be prepared from), to generate intermediate carrier DDD1-SV3.
Oligonucleotide primers shown in below using carries out pcr amplification to the hMN-14Fd sequence.
HMN-14VH left side G4S Bam
5’-GGATCCGGCGGAGGTGGCTCTGAGGTCCAACTGGTGGAGAGCGG-3’(SEQ?ID?NO：77)
CH1-C stops Eag
5’-CGGCCGTCAGCAGCTCTTAGGTTTCTTGTC-3’(SEQ?IDNO：78)
As the result of PCR, the coded sequence of the part of BamHI restriction enzyme site and connector promptly is connected to 5 ' end of amplimer.Termination codon and EagI restriction enzyme site then are connected to 3 ' end.This 1043bp amplimer is cloned into pGemT.Use BamHI and EagI restricted enzyme that the hMN-14-Fd insert is downcut from pGemT, then this insert 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 restricted enzyme that the N-DDD1-hMN-14Fd sequence is downcut, then this 1.28kb is inserted fragment and be connected to carrier segments through same enzyme digestion C-hMN-14-pdHL2 preparation.Final expression vector is N-DDD1-Fd-hMN-14-pDHL2.
The generation of embodiment 5.h679-Fab-AD1 and purification
679 antibodies HSG target antigens and affinity chromatograph capable of using carry out purification.The h679-Fd-ADl-pdHL2 carrier by linearisation, passes through electroporation method transfection Sp/EEE myeloma cell with the digestion of Sal1 restriction endonuclease then.Synthetic and the secretion of this two-cistron expression vector mediation h679 κ light chain and h679 Fd-AD1, they combine to form h679 Fab-AD1.After the electroporation, be coated with 96 hole tissue culturing plates, and select the transfection clone with 0.05 μ M methotrexate (MTX) with said cell.Use the protein expression of the microtitration plate of coating BSA-IMP-260 (HSG) conjugate, and use the goat anti human Fab who puts together HRP to detect through the ELISA screening and cloning.To use the BIAcore analysis of HSG (IMP-239) sensor chip to be used for confirming productive rate through measuring available from the initial slope of the diluted medium sample of injecting.The clone that output is the highest has the initial productive rate that is about 30mg/L.Use single step IMP-291 affinity chromatograph, from 4.5 liters of rolling bottle cultures, be purified into and amount to 230mg h679-Fab-AD1.Before adding to the IMP-291-affigel post, culture medium has concentrated about 10 times with hyperfiltration process.Use PBS that this post is eluted to baseline, use 1M imidazoles, 1mM EDTA, 0.1M NaAc, pH 4.5 eluting h679-Fab-AD1 then.The SE-HPLC of eluent analyzed show and have the single sharp peak of retention time (9.63 minutes) corresponding to 50kDa albumen (not shown).In reduction SDS-PAGE analyzes, the visible (not shown) of two bands of the polypeptide fractions of representing h679-AD1 is only arranged.
The generation of embodiment 6.N-DDD1-Fab-hMN-14 and C-DDD1-Fab-hMN-14 and purification
C-DDD1-Fd-hMN-14-pdHL2 and N-DDD1-Fd-hMN-14-pdHL2 carrier are gone into the deutero-myeloma cell of Sp2/0 through electroporation transfection.C-DDD1-Fd-hMN-14-pdHL2 is a two-cistron expression vector, the synthetic and secretion of its mediation hMN-14 κ light chain and hMN-14 Fd-DDD1, and they are combined to form C-DDD1-hMN-14 Fab.N-DDD1-hMN-14-pdHL2 is a two-cistron expression vector, the synthetic and secretion of its mediation hMN-14 κ light chain and N-DDD1-Fd-hMN-14, and they are combined to form N-DDD1-Fab-hMN-14.Each fusion rotein all forms stable homodimer through the interaction of DDD1 domain.
After the electroporation, be coated with 96 hole tissue culturing plates, and select the transfection clone with 0.05 μ M methotrexate (MTX) with said cell.Use the protein expression of the microtitration plate of coating WI2 (to the rat anti id monoclonal antibody of hMN-14), and use the goat anti human Fab who puts together HRP to detect through the ELISA screening and cloning.C-DDD1-Fab-hMN14Fab that output is the highest and N-DDD1-Fab-hMN14 Fab clone's initial productive rate is respectively 60mg/L and 6mg/L.
Use AD1-Affigel affinity purification N-DDD1-hMN-14 and C-DDD1-hMN-14
The construct that uses the DDD/AD interaction partners to comprise DDD1 carries out affinity purification.AD1-C is the peptide of being made up of ADI sequence and carboxyl terminal cysteine residues through synthetic preparation, and said cysteine residues is used for this peptide is coupled to Affigel (according to the reaction of sulfydryl and monochloroacetic acid anhydride).The a that comprises DDD 2Structure specificity under neutral pH combines the AD1-C-Affigel resin, and can be at eluting under the low pH (for example, pH 2.5).
Use single step AD1-C affinity chromatograph, from 1.2 liters of rolling bottle cultures, be purified into and amount to 81mg C-DDD1-Fab-hMN-14.Before adding to the AD1-C-affigel post, culture medium has concentrated about 10 times with hyperfiltration process.Use PBS that this post is eluted to baseline, use 0.1M glycine, pH 2.5 eluting C-DDD1-Fab-hMN-14 then.SE-HPLC analysis to eluent shows the single protein peak that exists retention time (8.7 minutes) consistent with 107kDa albumen (not shown).Purity only shows two band (not shown)s of the molecular size of two polypeptide fractions that are contemplated to C-DDD1-Fab-hMN-14 also through reduction SDS-PAGE analysis confirmation.
As stated, use single step ADI-C affinity chromatograph, from 1.2 liters of rolling bottle cultures, be purified into and amount to 10mg N-DDD1-hMN-14.SE-HPLC analysis to eluent shows that existence is similar to the retention time (8.77 minutes) of C-DDD1-Fab-hMN-14 and the single protein peak consistent with 107kDa albumen (not shown).Reduction SDS-PAGE analyzes and only shows two band (not shown)s corresponding to the polypeptide fractions of N-DDD1-Fab-hMN-14.
The active SE-HPLC through sample of the combination of C-DDD1-Fab-hMN-14 analyzes and confirms that in said sample, this trial target mixes with not commensurability WI2.The sample that WI2 Fab and C-DDD1-Fab-hMN-14 process by 0.75: 1 mixed in molar ratio shows three peaks, corresponding to C-DDD1-Fab-hMN14 (7.37 minutes) (not shown) of unconjugated C-DDD1-Fab-hMN14 (8.71 minutes), the C-DDD1-Fab-hMN-14 (7.95 minutes) that combines a WI2Fab and two WI2 Fab of combination.When WI2Fab that comprises when the sample of analyzing and the mol ratio of C-DDD1-Fab-hMN-14 are 4, only observe 7.36 minutes unimodal (not shown).These results prove that hMN14-Fab-DDD1 is a dimer, and two active binding sites are arranged.Use N-DDD1-Fab-hMN-14 to repeat this experiment, the result who obtains is closely similar.
Competitive ELISA proves that C-DDD1-Fab-hMN-14 all combines with CEA with the affinity similar with hMN-14 IgG with N-DDD1-Fab-hMN-14, and affinity significantly is better than unit price hMN-14 Fab (not shown).Use comprises the fusion rotein coating ELISA flat board of the special CEA epi-position (A3B3) of hMN-14.
Embodiment 7.a 2The formation of b complex
a 2The evidence that the b complex forms is by comprising equimolar C-DDD1-Fab-hMN-14 (as a 2) and the SE-HPLC of the mixture of h679-Fab-AD1 (as b) analyze and at first provide.When this sample is analyzed, observe retention time and be 8.40 minutes simple spike, this with formed greater than any independent h679-Fab-AD1 (9.55 minutes) or the consistent (not shown) of new albumen of C-DDD1-Fab-hMN-14 (8.73 minutes).As hMN-14 F (ab ') 2Mix with h679-Fab-AD1, when perhaps C-DDD1-Fab-hMN-14 mixes with 679-Fab-NEM, all do not observe this upfield shift, prove to interact clearly through DDD1 and the mediation of AD1 domain.Use h679-Fab-AD1 and N-DDD1-Fab-hMN-14 to obtain closely similar (not shown) as a result.
Use BIAcore to prove further and identified that the specificity between DD1 and the AD1 fusion rotein interacts.This experimentation is following: at first h679-Fab-AD1 or 679-Fab-NEM are attached to the surface of highdensity HSG link coupled (IMP239) sensor chip, inject C-DDD1-Fab-hMN-14 or hMN-14F (ab ') then 2Experimentize.As desired, when the injection latter, only there is h679-Fab-AD1 and C-DDD1-Fab-hMN-14 combination to cause the further increase (not shown) of response unit.Use h679-Fab-AD1 to obtain similar (not shown) as a result with C-DDD1-Fab-hMN-14.
Carry out balance SE-HPLC experiment and be present in AD1 and the interactional binding affinity of the specificity between the DDD1 in each fusion rotein with mensuration.Find the bonded dissociation constant (K of the commercial sample of h679-Fab-AD1 and C-DDD1-Fab-hMN-14, N-DDD1-hMN-14 and recombined human RII α d) be respectively 15nM, 8nM and 30nM.
The affinity purification of embodiment 8.DDD or AD fusion rotein
Have DDD that low-affinity more stops or AD albumen through generation and develop general affinity chromatograph system.The DDD that is formed by RI α dimer combines AKAP-IS (AD1) with 1/500 the affinity (225nM) of comparing with RII α.Therefore, can produce in the past RI α dimer that 44 amino acid residues form and it is coupled to resin is used for the fusion rotein of any AD1 of containing of purification with generation affinity substrate.
Nature exists many than low-affinity (0.1 μ M) AKAP anchoring structure territory.If desired, can introduce highly predictable amino acid replacement with the said binding affinity of further reduction.Low-affinity AD can prepare through synthetic or biological method, and can be coupled to resin to be used for the affinity purification of any DDD1 fusion rotein.
Embodiment 9. is used to produce the carrier of disulfide bond rock-steady structure
N-DDD2-Fd-hMN-14-pdHL2
N-DDD2-hMN-14-pdHL2 is the expression vector that is used to produce N-DDD2-Fab-hMN-14, and it has the dimerization and stop domain sequence of the DDD2 that is connected with the amino terminal of Fd.DDD2 is coupled to V through the Gly/Ser peptide connector of 15 amino acid residues HDomain.DDD2 has the dimerization sequence identical with the sequence of DDD1 and stops sequence, but before them, has cysteine residues.
As follows said expression vector is transformed.The eclipsed complementary oligonucleotide (DDD2 last and DDD2 under) that comprises the 1-13 position residue of DDD2 through two of synthetic preparations.Said oligonucleotide is annealed, and with T4 polynucleotide kinase (PNK) phosphorylation, be fit to overhang with the DNA that digests with restriction endonuclease NcoI and PstI respectively is connected to form at 5 ' and 3 ' end.
DDD2 is last
5’CATGTGCGGCCACATCCAGATCCCGCCGGGGCTCACGGAGCTGCTGCA-3’(SEQ?ID?NO：79)
Under the DDD2
5’GCAGCTCCGTGAGCCCCGGCGGGATCTGGATGTGGCCGCA-3’(SEQ?ID?NO：80)
Said duplex DNA is connected with carrier segments DDD1-hMN14 Fd-SV3 (through with NcoI and PstI digestion preparation), with construct DDD2-hMN14 Fd-SV3 in the middle of generating.Use XhoI and EagI restriction endonuclease, the 1.28kb that will comprise DDD2-hMN14 Fd coded sequence inserts fragment and downcuts from said middle construct, then this fragment is connected to the hMN14-pdHL2 carrier DNA that uses 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 that is used to produce C-DDD2-Fab-hMN-14, its have Gly/Ser peptide connector through 14 amino acid residues be connected to Fd carboxyl terminal DDD2 dimerization with stop the domain sequence.As follows said expression vector is transformed.The eclipsed complementary oligonucleotide of 1-13 position residue that comprises coded sequence (GGGGSGGGCG, SEQ ID NO:81) and the DDD2 of the part of connector peptide through two of synthetic preparations.With the annealing of said oligonucleotide, and with T4 PNK phosphorylation, suitablely overhang with the DNA that digests with restriction endonuclease BamHI and PstI respectively is connected to form with 3 ' end 5 '.
G4S-DDD2 is last
5’GATCCGGAGGTGGCGGGTCTGGCGGAGGTTGCGGCCACATCCAGATCCCGCCGGGGCTCACGGAGCTGCTGCA-3’(SEQ?IDNO：82)
Under the G4S-DDD2
5’GCAGCTCCGTGAGCCCCGGCGGGATCTGGATGTGGCCGCAACCTCCGCCAGACCCGCCACCTCCG-3’(SEQ?ID?NO：83)
Said double-stranded DNA is connected with shuttle vector CH1-DDD1-pGemT (continuous and meandering is crossed with BamHI and PstI digestion preparation), to generate shuttle vector CH1-DDD2-pGemT.Use SacII and EagI that the 507bp fragment is downcut from CH1-DDD2-pGemT, then this fragment is connected to IgG expression vector hMN14 (the I)-pdHL2 that uses 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 that is used to produce h679-Fab-AD2, and it has the anchoring structure territory sequence of AD2 that Gly/Ser peptide connector through 14 amino acid residues is connected to the carboxyl terminal of CH1.Respectively there is a cysteine residues front and back of AD2 sequence in AD1 anchoring structure territory.
As follows said expression vector is transformed.Comprise the coded sequence of AD2 and the eclipsed complementary oligonucleotide of part connector sequence (AD2 last and AD2 under) through two of synthetic preparations.With the annealing of said oligonucleotide, and with T4 PNK phosphorylation, form with 3 ' end 5 ' and suitablely to overhang with the DNA that digests with restriction endonuclease BamHI and SpeI respectively is connected.
AD2 is last
5’GATCCGGAGGTGGCGGGTCTGGCGGATGTGGCCAGATCGAGTACCTGGCCAAGCAGATCGTGGACAACGCCATCCAGCAGGCCGGCTGCTGAA-3’(SEQ?ID?NO：84)
Under the AD2
5’TTCAGCAGCCGGCCTGCTGGATGGCGTTGTCCACGATCTGCTTGGCCAGGTACTCGATCTGGCCACATCCGCCAGACCCGCCACCTCCG-3’(SEQ?ID?NO：85)
Said duplex DNA is connected with shuttle vector CH1-AD1-pGemT (through with BamHI and Spel digestion preparation), to generate shuttle vector CH1-AD2-pGemT.The 429 base pair fragments of using SacII and EagI restricted enzyme will comprise CH1 and AD2 coded sequence are downcut from said shuttle vector, then this fragment are connected to the h679-pdHL2 carrier that uses same enzyme digestion preparation.Final expression vector is h679-Fd-AD2-pdHL2.
The generation of embodiment 10.TF1
Carry out the DNL construct mass preparation of (being called TF1) as follows.At first according to roughly stoichiometric concentration with N-DDD2-Fab-hMN-14 (albumen L-purification) and h679-Fab-AD2 (the IMP-291-purification) at 1mM EDTA, PBS mixes among the pH 7.4.Add before the TCEP, SE-HPLC does not demonstrate a 2Any evidence (not shown) that b forms.But has representative a 4(7.97 minutes; 200kDa), a 2(8.91 minutes; 100kDa) and B (10.01 minutes; Peak 50kDa).Add 5mM TCEP and cause a fast 2The formation of b complex, consistent with 150kDa albumen, proved this point (not shown) at 8.43 minutes new peak.Excessive B is obviously arranged in this experiment, because be still significantly corresponding to the peak of h679-Fab-AD2 (9.72 minutes), but corresponding to a 2Or a 4Then do not see tangible peak.After the reduction reaction 1 hour, carry out dialysed overnight, remove TCEP to the PBS that changes several times.Gained solution adds 10%DMSO and at room temperature preserves and spend the night.
When analyzing, represent a with SE-HPLC 2It is sharper that the peak of b obviously becomes, and retention time has reduced by 0.1 minute a little, becomes 8.31 minutes (not shown)s, according to our previous discovery, and the raising of this expression binding affinity.This complex is further purified to remove κ chain impurity through the IMP-291 affinity chromatograph.As desired, excessive h679-AD2 is come out by purification together, removes (not shown) with preparation type SE-HPLC then.
TF1 is high stability complex.When measuring TF1 and HSG (IMP-239) when sensor chip combines; Obvious reduction is not seen in observed response after the sample injection finishes; By contrast, when the solution of C-DDD1-Fab-hMN-14 that under condition of similarity, measures molar mixtures such as containing and h679-Fab-AD1, during the sample injection and after firm the injection; The increase of viewed response unit is accompanied by detectable decline, shows a that begins to form 2The b structural instability.In addition, although inject WI2 subsequently, for the C-DDD1/AD1 mixture, obviously do not increase increasing to causing in the response unit of TF1 significantly.
The extra increase of the response unit that combines with TF1 on being fixed on sensor chip because of WI2 to cause, corresponding to two complete functional binding sites, its each cause by the subunit of N-DDD2-Fab-hMN-14 naturally.Two segmental binding abilities of Fab of TF1 and WI2 have confirmed this point (not shown).After the mixture that contains h679-AD2 and N-DDD1-hMN14 was correctly reduced and is oxidized into TF1, when analyzing with BIAcore, WI2 did not almost have extra combination (not shown), shows a that disulfide bond is stable 2B complex (for example TF1) the only interaction through DDD2 and AD2 forms.
This process two improvement have been carried out, to reduce the time and the efficient of process.At first, with a 4/ a 2The N-DDD2-Fab-hMN-14 of the molar excess a little that the structure form of mixtures exists and h679-Fab-AD2 reaction, making does not have free h679-Fab-AD2 to exist, and is not bound to any a of h679-Fab-AD2 4/ a 2Structure and light chain all use the IMP-291 affinity chromatograph to remove.The second, hydrophobic interaction chromatography (HIC) replaces dialysis or diafiltration, after reduction reaction, is used to remove TCEP, and this not only shortens process time, but also has increased the removal step of potential virus.N-DDD2-Fab-hMN-14 mixes with 679-Fab-AD2, at room temperature reduces 1 hour with 5mM TCEP.Solution adds 0.75M ammonium sulfate, goes up appearance then to butyl FF HIC post.PBS with containing 0.75M ammonium sulfate, 5mM EDTA washes post, to remove TCEP.With PBS eluting reduction albumen from the HIC post, add 10%DMSO.At room temperature after the incubated overnight, isolate highly purified TF1 (Figure 22) with the IMP-291 affinity chromatograph.Need not additional purification step, example gel filters.
The generation of embodiment 11.TF2
Success produces after the TF1, with C-DDD2-Fab-hMN-14 and h679-Fab-AD2 reaction, also obtains a kind of analog, is called TF2.The following TF2 that produces with the pilot batch that surpasses 90% yield.C-DDD2-Fab-hMN-14 (200mg) and the mixed in molar ratio of h679-Fab-AD2 (60mg) with albumen L-purification with 1.4: 1.In the PBS that contains 1mM EDTA, total protein concentration is 1.5mg/ml.Subsequent step comprises TCEP reduction, HIC chromatography, DMSO oxidation and IMP-291 affinity chromatograph, all with described identical to TF1.Add before the TCEP, SE-HPLC does not demonstrate a 2Any evidence (not shown) that b forms.But exist corresponding to a 4(8.40 minutes; 215kDa), a 2(9.32 minutes; 107kDa) and b (10.33 minutes; Peak 50kDa).Add 5mM TCEP and cause a fast 2The formation of b complex, as 8.77 minutes (not shown) new peak proved, this peak is consistent with the desired 157kDa albumen of diadactic structure.With the IMP-291 affinity chromatograph TF2 is purified near the homogenizing (not shown).SE-HPLC analytical proof to IMP-291 does not combine fraction to carry out has been removed a from product 4, a 2With free κ chain (not shown).
According to for the described function of utilizing BIACORE to measure TF2 of TF1.TF2, C-DDD1-hMN-14+h679-AD1 (are used as non-covalent a 2The control sample of b complex) or C-DDD2-hMN-14+h679-AD2 (as non-reduced a 2Control sample with the b component) is diluted to 1 μ g/ml (gross protein) and through having fixed the sensor chip of HSG.To the response of TF2 is about 2 times of two control samples reactions, and showing only has the h679-Fab-AD component to combine with sensor chip and keep above that in the control sample.Inject WI2IgG subsequently, prove the DDD-Fab-hMN-14 component that only has TF2 to have to combine closely, shown in extra signal response with h679-Fab-AD.The response unit that combines with TF2 on being fixed on sensor chip to cause because of WI2 increases, and also corresponding to two complete functional binding sites, each all is that a subunit by C-DDD2-Fab-hMN-14 causes.Two segmental binding abilities of Fab of TF2 and WI2 have confirmed this point (not shown).
Measure the relative CEA-binding affinity of TF2 through competitive ELISA.Fusion rotein with the A3B3 domain that contains CEA encapsulates each plate (0.5 μ g/ hole), and said albumen is discerned by hMN-14.Serial dilution TF1, TF2 and hMN-14IgG, quadruplicate, incubation in the hole of containing the hMN-14IgG (1nM) that puts together HRP.Data show TF2 combines CEA with the affinity that equates at least with the affinity of IgG, than the strong 2 times of (not shown)s of TF1.
The serum stability of embodiment 12.TF1 and TF2
TF1 and TF2 are designed to can be used for intravital stable constraint structure, wherein in blood and tissue, Macrodilution can take place.With the stability of TF2 in the BIACORE evaluator serum, TF2 is diluted to 0.1mg/ml with Freshman serum (merge and obtain from 4 donors) and at 5%CO 2, 37 ℃ of incubations 7 days.Every day, sample diluted by 1: 25, analyzed through BIACORE then, used the IMP-239HSG sensor chip.Inject WI2IgG, be used for the amount of the complete and complete active TF2 of quantitative assay.Blood serum sample is compared with the control sample that directly dilutes from stock solution.TF2 is stable at the serum camber, and its bispecific of maintenance 98% combines active (not shown) after 7 days.In people or mice serum, observe similar (not shown) as a result for TF1.
The generation of embodiment 13.C-H-AD2-IgG-pdHL2 expression vector
The pdHL2 mammalian expression vector has been used to mediate the expression of many reorganization IgG, and (people such as Qu, Methods 2005,36:84-95).Producing the plasmid shuttle vector is beneficial to any IgG-pdHL2 carrier is converted into the C-H-AD2-IgG-pdHL2 carrier.Use the pdHL2 carrier as template and an oligonucleotide Fc BglII left side and the Fc Bam-EcoRI right side gene as primer amplification Fc (CH2 and CH3 domain).
A Fc BglII left side
5’-AGATCTGGCGCACCTGAACTCCTG-3’(SEQ?ID?NO：86)
Fc Bam-EcoRI is right
5’-GAATTCGGATCCTTTACCCGGAGACAGGGAGAG-3’(SEQID?NO：87)
Amplimer is cloned into pGemT PCR cloning vehicle.Use XbaI and BamHI restricted enzyme to downcut Fc insert fragment, be connected to then with XbaI and BamHI and digest h679-Fab-AD2-pdHL2 and the AD2-pdHL2 carrier for preparing, with generation shuttle vector Fc-AD2-pdHL2 from pGemT.
For any IgG-pdHL2 expression vector is changed into the C-H-AD2-IgG-pdHL2 expression vector, downcut 861bp BsrGI/NdeI restriction fragment from the former, it is replaced with the 952bp BsrGI/NdeI restriction fragment that downcuts from the Fc-AD2-pdHL2 carrier.BsrGI cuts in the downstream (3 ') of expression cassette in CH3 domain incised and NdeI.
The generation of embodiment 14.C-H-AD2-hLL2 IgG
Epratuzumab (or hLL2 IgG) is humanization Anti-Human CD22 MAb.As stated, from the expression vector that hLL2 IgG-pdHL2 produces C-H-AD2-hLL2 IgG, use it for transfection Sp2/0 myeloma cell through electroporation.After the transfection, be coated with 96 orifice plates, in containing the culture medium of methotrexate, select transgene clone with said cell.The sandwich ELISA of the microtitration plate through using coating hLL2-specific anti-idiotype MAb and the C-H-AD2-hLL2 IgG productive rate of detection screening and cloning of puting together the Anti-Human IgG of peroxidase.With clonal expansion to rolling bottle with preparation protein, and in using the single stage of protein-A affinity chromatograph the culture medium purification C-H-AD2-hLL2 IgG from exhausting.SE-HPLC analyzes two protein peaks is resolved (not shown).The retention time (8.63 minutes) of slow eluting peak is similar with hLL2IgG.The retention time (7.75 minutes) of fast eluting peak is corresponding to about 300kDa protein.On behalf of the disulfide bond of C-H-AD2-hLL2-IgG, definite afterwards this peak connect dimer.This dimer is reduced to monomeric form in the DNL reaction.SDS-PAGE analyzes and to show, the C-H-AD2-hLL2-IgG of purification forms (not shown) by the dimeric forms that two kinds of monomers of module are connected with disulfide bond.Represent among the SDS-PAGE of protein band under the irreducibility condition of these two kinds of forms high-visible, but under reducing condition, form of ownership all is reduced to two band (not shown)s representing composition polypeptide (heavy chain-AD2 and κ chain).Do not detect other and pollute band.
The generation of embodiment 15.C-H-AD2-hA20 IgG
HA20 IgG is humanization anti-humen CD 20 MAb.Expression vector from hA20 IgG-pDHL2 generation C-H-AD2-hA20 IgG passes through electroporation transfection Sp2/0 myeloma cell with this carrier then.After the transfection, be coated with 96 orifice plates, and screen transgene clone comprising on the culture medium of methotrexate with said cell.The C-H-AD2-hA20 IgG productive rate of the sandwich ELISA of 96 hole microtitration plates through using coating hA20 specific anti idiotype MAb and the detection screening and cloning of the anti-human IgG that peroxidase is puted together in use.With clonal expansion to rolling bottle with the preparation protein; And in using the single stage of protein-A affinity chromatograph the culture medium purification C-H-AD2-hA20 IgG from exhausting, SE-HPLC and SDS-PAGE analyze the result that obtains and the closely similar (not shown) of result for C-H-AD2-hLL2 IgG acquisition.
The generation that embodiment 16.AD-is connected with DDD-from the Fab and the IgG fusion rotein of a plurality of antibody
Through using the technology of describing in the previous embodiment, make up following IgG or Fab fusion rotein, and be integrated into the DNL construct.The antibody that the antigen combination characteristic of said fusion rotein maintenance parental generation antibody and the demonstration of DNL construct are integrated or the antigen-binding activity of antibody fragment.
Table 3. 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 17. comprises the DNL immunotoxin based on ribonuclease of the 4 times of body ranpirnases (Rap) that are conjugated to the B cell lymphoma targeting antibodies
We use stop-produce the immunotoxin of novel kind with-(DNL) method that locks, and the locus specificity of its each self-contained 4 copies is connected to the Rap of bivalence IgG.We with the reorganization Rap-DDD module that produces in the escherichia coli (E.coli) and recombinant humanized IgG-AD module (it produces in the myeloma cell and targeting B cell lymphoma and leukemia) through with CD20 (hA20; Veltuzumab), CD22 (hLL2; Epratuzumab) or HLA-DR (hL243; IMMU-114) combine to combine, to produce 20-Rap, 22-Rap and C2-Rap respectively.For each construct, the dimer of Rap is by the C of covalent tethering to each heavy chain of IgG separately end.Labetuzumab (hMN-14) the preparation contrast construct 14-Rap of the antigen (CEACAM5) that use to combine similarly on B cell lymphoma/leukemia, not express.
pQDWLTFQKKHITNTRDVDCDNIMSTNLFHCKDKNTFIYSRP EPVKAICKGIIASKNVLTTSEFYLSDCNVTSRPCKYKLKKSTNKFC VTCENQAPVHFVGVGSC
Figure BDA0000139409280000913
HHHHHH(SEQ?ID?NO：94)
The deduced amino acid of secreting type Rap-DDD2 is shown in top (SEQ IDNO:94).Rap, underlined; Connector, italics; DDD2, runic; PQ is transformed into the amino terminal glutamine of pyroglutamic acid.Rap-DDD2 is produced as Inclusion in escherichia coli, through the said Inclusion of IMAC purification, refolding is being dialysed to PBS through before the Q-agarose anion-exchange chromatography purification then under the degeneration condition.SDS-PAGE under the reducing condition parses the protein band (not shown) that has corresponding to the Mr of Rap-DDD2 (18.6kDa).The ultimate yield of the Rap-DDD2 of purification is the 10mg/L culture.
The DNL method is used for producing fast one group of IgG-Rap conjugate.The IgG-AD module is expressed in the myeloma cell, used the protein A affinity chromatograph that its purification from culture supernatant is come out then.Produce the Rap-DDD2 module, it is mixed with IgG-AD2 to form the DNL complex.Because the CH3-AD2-IgG module has 2 AD2 peptides and each can fetter the Rap dimer, so the IgG-Rap DNL construct of gained comprises 4 Rap groups and 1 IgG.IgG-Rap almost forms and is purified near homogenizing by protein A from forming module quantitatively.
Before the DNL reaction, CH3-AD2-IgG exists with the dimer (not shown) that monomer is connected with disulfide bond.Under the irreducibility condition, IgG-Rap resolves to the HMW band (not shown) of the expection size of cluster between monomer and dimer CH3-AD2-IgG.The reductive condition that conjugate is reduced to their composition polypeptide demonstrates the purity of IgG-Rap and the concordance of DNL method, because only show the band (not shown) of representative heavy chain-AD2 (HC-AD2), κ light chain and Rap-DDD2.
The reversed-phase HPLC of 22-Rap is analyzed (not shown) parse the single protein peak at 9.10 minutes eluting, it is between two peaks (representing monomeric form (7.55 minutes) and dimeric forms (8.00 minutes)) of CH3-AD2-IgG-hLL2.The Rap-DDD2 module is separated with the form of mixtures of the dimer and the tetramer (in DNL, being reduced into dimer), and it respectively can be 9.30 minutes and 9.55 minutes by the eluting (not shown).
LC/MS analysis to 22-Rap realizes (not shown) through reversed-phase HPLC and the coupling of ESI-TOF mass spectrography that will use the C8 pillar.The mass spectrum of adorned 22-Rap has not been identified two main kinds, and except some less important sugar form (not shown)s, it also has the polysaccharide of two G0F (G0F/G0F) or a G1F of G0F+ (G0F/G1F) N-connection.The enzymatic deglycosylation causes the single overlapping combination mass spectrum (not shown) that conforms to the quality through calculating of 22-Rap.At heavy chain-AD2 polypeptide of having identified that with the mass spectrum of TCEP reduction back gained the polysaccharide that connected by the N-of G0F or G1F structure and other less important form (not shown) are modified.Each of 3 subunit polypeptide that comprises 22-Rap obtains identifying (not shown) in the overlapping combination spectrum that is reduced with deglycosylated sample.Said results verification Rap-DDD2 and HC-AD2 polypeptide all have the amino terminal glutamine that is converted into pyroglutamic acid (pQ); Thereby 22-Rap has its 8 by 6 polypeptide in the composition polypeptide of pQ modification.
In 3 kinds of NHL cell lines, assess vitro cytotoxicity.Each cell line is expressed CD20 with the area density of comparing much higher with CD22; Yet (anti--internalization speed CD22) is far faster than hA20 (anti-CD 20) for hLL2.14-Rap and 22-Rap and the total identical structure of 20-Rap, but its antigen (CEACAM5) is not by the NHL cellular expression.In Figure 13, left figure: handle cell continuously as single agents or with the combination of parental generation MAb+rRap with IgG-Rap.20-Rap and 22-Rap kill and wound each cell line being higher than on the concentration of 1nM, this shows their effect and only the cell growth inhibited is opposite, has cytotoxicity.20-Rap is effective I gG-Rap, shows that antigen density maybe be more important than internalization speed.Obtain similar result, 20-Rap (EC in said cell for Daudi with Ramos 50About 0.1nM) effectiveness is 3-6 times of 22-Rap.Anti-Rituximab mantle cell lymphoma strain Jeko-1 compares with Daudi and Ramos and shows the CD20 that increases but the CD22 that reduces.Important ground, 20-Rap shows extremely strong cytotoxicity (EC in Jeko-1 50About 20pM), it renders a service 25 times for 22-Rap.
Like what show among Figure 13, right figure: among the expectation is to reduce the cytotoxicity (about 50 times) of each reagent significantly at 1 hour processing after scouring cell.Again, 20-Rap is the most effective, shows that its slower internalization speed is not determinate.14-Rap compares with rRap (with the combination of MAb) and shows the cytotoxicity that increases, and these 4 times of Rap structures that show gG-Rap can increase its internalization.The cytotoxicity that incubation after scouring at 1 hour makes 14-Rap than falling of targeting 22-Rap and 20-Rap more.
Use 3 ALL cell line assessment IgG-Rap (Figure 14).Antigen density is similar relatively between 3 cell lines, HLA-DR＞＞CD22＞CD20.There is not a kind of parental generation MAb (making up individually or with rRap) in these are measured, to have cytotoxicity.Yet non-targeting 14-Rap shows certain activity, and is similar with the result of NHL strain.For each cell line, the C2-Rap of the antigen (HLA-DR) that targeting is the abundantest produces the most effectively reaction, and it is about 50 times of 22-Rap.For ALL strain with extremely low CD20 antigen density, the cytotoxicity of 20-Rap showed moderate, this cytotoxicity is similar with the cytotoxicity of non-targeting 14-Rap.This with have high CD20 density and opposite to the result of the strongest NHL strain of 20-Rap reaction.Therefore, the effect of IgG-Rap is relevant with the relative abundance of targeting antigen.
Conclusion
The DNL method provides the modular method that efficiently a plurality of cytotoxins is bound to (thereby causing expecting that pharmacokinetics and targeting specific because of improving show the novel immunotoxin of rendeing a service in the higher body) on the targeting antibodies.LC/MS, RP-HPLC and SDS-PAGE have proved homogeneity and the purity of IgG-Rap.Utilize MAb that Rap targeted cells surface antigen has been strengthened its tumor-specific cytotoxicity.Antigen density and internalization speed are two vital factors of the vitro efficacy of observed IgG-Rap.In vitro results shows that CD20-, CD22-or HLA-DR-targeting IgG-Rap have the effective biological activity that is used for B cell lymphoma and leukemic treatment.
Embodiment 18.Rap-resists-the anticancer efficient of Trop-2 IgG DNL construct
Through using describe among the embodiment 17 constructed; Generation comprises the hRS7-IgG-Ad2 of the Rap-DDD2 that is connected to 4 copies, and (anti--Trop-2) E1-Rap DNL construct, said construct has shown the effective growth in vitro rejection characteristic (not shown) of anti-multiple cancerous cell line.In mammary gland (MDA-MB-468), cervix uteri (ME-180) and pancreas (BxPC-3 and Capan-1) tumor strain, it all expresses high-caliber Trop-2, and E1-Rap renders a service very high, has shown the EC in inferior nanomole (subnanomolar) scope (5 to 890pM) 50, it is 1,000 times to 100,000 times of combination of non-targeting Rap or Rap and hRS7.For example in 3 carcinoma of prostate strains (PC-3, DU 145 and LNCaP), E1-Rap renders a service not too high, but still is presented at the EC in the nanomole scope (1 to 890nM) in the cell line of the Trop-2 that expresses medium level 50As if the cell about these solid carcinoma cells that obtains combine tables of data clear-cells system to express relevant with its Trop-2 on cell surface to the sensitivity of E1-Rap.In can not combining the carcinoma of prostate strain 22Rv1 of hRS7, do not observe the toxicity of E1-Rap.These results have shown the effect of E1-Rap as the new therapeutic agent of the positive solid tumor of Trop-2-(comprising breast carcinoma, colon cancer, gastric cancer, pulmonary carcinoma, ovarian cancer, carcinoma of endometrium, cervical cancer, cancer of pancreas and carcinoma of prostate).
***
Can and use all compositionss and the method with the requirement protection disclosed herein to need not undo experimentation according to the present disclosure preparation.Though described compositions and method through embodiment preferred, it should be apparent that to those skilled in the art and can change and do not deviate from notion of the present invention, spirit and scope to the order of compositions and method or the method for describing in this article or step.More specifically, some all relevant reagent can be used for the reagent of describing among alternative this paper and can obtain same or analogous result on chemistry and physiology.It should be apparent that to those skilled in the art all this type of similar substitute and modifications be considered to as by accompanying claims in determined spirit of the present invention, scope and the notion.

Claims (45)

1. a DNL (stop and lock) construct, it comprises:
A) be connected to the toxin of DDD from PKA (PKA) (dimerization with stop domain) part; With
B) be connected to antibody or antigen binding antibody fragment partly from the proteic AD of AKAP (anchoring structure territory);
Wherein the DDD of two copies partly forms dimer and partly combines to form said DNL construct with AD.
2. the described DNL construct of claim 1, wherein said antibody or antibody fragment comprise the AD part of two copies.
3. the described DNL construct of claim 2, wherein said AD partly is connected to the C-terminal of the heavy chain of said antibody or antibody fragment.
4. the described DNL construct of claim 2, wherein said DNL construct comprises the toxin of 4 copies.
5. the described DNL construct of claim 1, wherein said toxin is selected from bacteriotoxin, phytotoxin, ricin, Agglutinin, alpha toxin, saporin, ribonuclease (RNA enzyme), DNA enzyme I, staphylococcus enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, diphtherotoxin, PE, pseudomonas endotoxin, ranpirnase (Rap) and Rap (N69Q).
6. the described DNL construct of claim 5, wherein said toxin is ranpirnase (Rap).
7. the described DNL construct of claim 1; Wherein said antibody or antibody fragment conjugated antigen, said antigen are selected from ED-B, 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, neoplasm necrosis antigen, TNF-α, TRAIL receptor (R1 and R2), VEGFR, EGFR, PlGF, complement factor C3, C3a, C3b, C5a, C5 and the oncoprotein of 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, fibronectin.
8. the described DNL construct of claim 1, wherein said antibody or antibody fragment be selected from hR1 (anti--IGF-1R), hPAM4 (anti--mucin), 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) with hMN-3 (resist-CEACAM6).
9. the described DNL construct of claim 7, wherein said antibody or antibody fragment for anti--EGP-1 (anti--Trop-2), anti--CD74, anti--CD22 or anti-CD 20.
10. the described DNL construct of claim 9, wherein said antibody or antibody fragment be selected from hRS7 (anti--Trop-2), milatuzumab (anti--CD74), veltuzumab (anti--CD22) and epratuzumab (anti-CD 20).
11. the described DNL construct of claim 1, wherein said DDD partly have the aminoacid sequence from people RI α, RI β, RII α or RII β PKA.
12. the described DNL construct of claim 1, wherein said toxin and antibody or antibody fragment are fusion rotein, and each fusion rotein comprises AD or DDD part.
13. the described DNL construct of claim 1, wherein said DDD partly have preceding 44 aminoacid of preceding 44 aminoacid, SEQ ID NO:21, SEQ IDNO:22 or SEQ ID NO:59 of aminoacid sequence, the SEQ ID NO:20 of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:18.
14. the described DNL construct of claim 1, wherein said AD partly have the aminoacid sequence of SEQID NO:15, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ IDNO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ IDNO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ IDNO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, M SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ IDNO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ IDNO:56, SEQ ID NO:57 or SEQ ID NO:58.
15. the described DNL construct of claim 1, wherein said antibody or antibody fragment are selected from IgG, F (ab ') 2, F (ab) 2, Fab ', Fab, Fv, sFv, scFv and dAb.
16. a method of using toxin to the experimenter comprises to the experimenter and uses DNL construct according to claim 1.
17. the described method of claim 16; Wherein said antibody or antibody fragment conjugated antigen, said antigen are selected from ED-B, 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, neoplasm necrosis antigen, TNF-α, TRAIL receptor (R1 and R2), VEGFR, EGFR, PlGF, complement factor C3, C3a, C3b, C5a, C5 and the oncoprotein of 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, fibronectin.
18. the described method of claim 17, wherein said antibody or antibody fragment are T anti--EGP-1 (anti--Trop-2), anti--CD74, anti--CD22 or anti-CD 20.
19. the described method of claim 16, wherein said antibody or antibody fragment be selected from hR1 (anti--IGF-1R) hPAM4 (anti--mucin), 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) with hMN-3 (resist-CEA).
20. the described method of claim 16, wherein said antibody or antibody fragment be selected from hRS7 (anti--Trop-2), milatuzumab (anti--CD74), veltuzumab (anti--CD22) and epratuzumab (anti-CD 20).
21. the described method of claim 16, wherein said toxin are selected from bacteriotoxin, phytotoxin, ricin, Agglutinin, alpha toxin, saporin, ribonuclease (RNA enzyme), DNA enzyme I, staphylococcus enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, diphtherotoxin, PE, pseudomonas endotoxin, ranpirnase (Rap) and Rap (N69Q).
22. the described method of claim 16, wherein said toxin are ranpirnase (Rap).
23. a treatment is selected from the method for the disease of cancer, immune dysfunction and autoimmune disease, comprises to the experimenter who suffers from said disease using DNL construct according to claim 1.
24. the described method of claim 23, wherein said cancer are selected from non_hodgkin lymphoma, B cell lymphoma, B cell leukemia, t cell lymphoma, T HTLV, acute lymphoblastic appearance leukemia, chronic lymphoid leukemia, Burkitt lymphoma, He Jiejin lymphomas, hairy cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, multiple myeloma, glioma, Walden Si Telun macroglobulinemia, cancer, melanoma, sarcoma, glioma, skin carcinoma, oral cancer, gastrointestinal cancer, colon cancer, gastric cancer, lung road cancer, pulmonary carcinoma, breast carcinoma, ovarian cancer, carcinoma of prostate, uterus carcinoma, endometrium cancer, cervical cancer, bladder cancer, cancer of pancreas, osteocarcinoma, hepatocarcinoma, carcinoma of gallbladder, renal carcinoma and carcinoma of testis.
25. the described method of claim 23, wherein said autoimmune disease are selected from nephritis, 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, 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, tabes dorsalis, giant cell arteritis/polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis and fibrosing alveolitis after acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus (sle), lupus nephritis, rheumatic fever, polyglandular syndrome, bullous pemphigoid, diabetes, purpura,Henoch-Schonlein, the streptococcal infection.
26. the described method of claim 23; Wherein said antibody or antibody fragment conjugated antigen, said antigen are selected from ED-B, 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, neoplasm necrosis antigen, TNF-α, TRAIL receptor (R1 and R2), VEGFR, EGFR, PlGF, complement factor C3, C3a, C3b, C5a, C5 and the oncoprotein of 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, fibronectin.
27. the described method of claim 23, wherein said antibody or antibody fragment be selected from hR1 (anti--IGF-1R) hPAM4 (anti--mucin), 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) with hMN-3 (resist-CEA).
28. the described DNL construct of claim 23, wherein said antibody or antibody fragment be selected from hRS7 (anti--Trop-2), milatuzumab (anti--CD74), veltuzumab (anti--CD22) and epratuzumab (anti-CD 20).
29. the described method of claim 23, wherein said toxin are selected from bacteriotoxin, phytotoxin, ricin, Agglutinin, alpha toxin, saporin, ribonuclease (RNA enzyme), DNA enzyme I, staphylococcus enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, diphtherotoxin, PE, pseudomonas endotoxin, ranpirnase (Rap) and Rap (N69Q).
30. the described method of claim 29, wherein said toxin are ranpirnase (Rap).
31. one kind comprises the immunotoxin that is connected to antibody or the segmental ranpirnase of antigen binding antibody.
32. the described immunotoxin of claim 31; Wherein said immunotoxin has structure 2L-Rap (Q)-MAb, wherein Rap (Q) be the mutant form of the ranpirnase (Rap) that is removed of the N-glycosylation site wherein supposed and wherein MAb be antibody or antigen binding antibody fragment.
33. the described immunotoxin of claim 31, wherein said antibody or antibody fragment be anti--Trop-2 (anti--EGP-1).
Resist-Trop-2 antibody 34. the described immunotoxin of claim 33, wherein said antibody or antibody fragment are humanizations, it comprises heavy chain CDR sequence C DR1 (NYGMN; SEQID NO:1), CDR2 (WINTYTGEPTYTDDFKG; SEQ ID NO:2) and CDR3 (GGFGSSYWYFDV, SEQ ID NO:3) and light chain CDR sequence C DR1 (KASQDVSIAVA, SEQ ID NO:4), CDR2 (SASYRYT; SEQ IDNO:5) and CDR3 (QQHYITPLT, SEQ ID NO:6).
35. the described immunotoxin of claim 33, wherein said immunotoxin has cytotoxicity to cancerous cell line on 100nM or lower concentration.
36. the described immunotoxin of claim 35, wherein said cancerous cell line are cervical cancer, breast carcinoma, carcinoma of prostate, adenocarcinoma of lung, cancer of pancreas or ovarian cancer cell line.
37. the described immunotoxin of claim 33, wherein said immunotoxin has cytotoxicity to cancerous cell line on 10nM or lower concentration.
38. the described immunotoxin of claim 33, wherein immunotoxin is the fusion rotein that comprises the Rap (Q) of the VL sequence of N end that is connected to antibody or antibody fragment.
39. a treatment is selected from the method for the disease of cancer, immune dysfunction and autoimmune disease, comprises to the experimenter who suffers from said disease using immunotoxin according to claim 32.
40. the described method of claim 39; Wherein said antibody or antibody fragment conjugated antigen, said antigen are selected from ED-B, 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, neoplasm necrosis antigen, TNF-α, TRAIL receptor (R1 and R2), VEGFR, EGFR, PlGF, complement factor C3, C3a, C3b, C5a, C5 and the oncoprotein of 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, fibronectin.
41. the described method of claim 39, wherein said antibody or antibody fragment be selected from hR1 (anti--IGF-1R), hPAM4 (anti--mucin), 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) with hMN-3 (resist-CEACAM6).
42. the described method of claim 39, wherein said antibody or antibody fragment be anti--Trop-2 (anti--EGP-1).
Resist-Trop-2 antibody 43. the described method of claim 42, wherein said antibody or antibody fragment are humanizations, it comprises heavy chain CDR sequence C DR1 (NYGMN; SEQ IDNO:1), CDR2 (WINTYTGEPTYTDDFKG; SEQ ID NO:2) and CDR3 (GGFGSSYWYFDV, SEQ ID NO:3) and light chain CDR sequence C DR1 (KASQDVSIAVA, SEQ ID NO:4), CDR2 (SASYRYT; SEQ IDNO:5) and CDR3 (QQHYITPLT, SEQ ID NO:6).
44. the described method of claim 39, wherein said cancer are selected from non_hodgkin lymphoma, B cell lymphoma, B cell leukemia, t cell lymphoma, T HTLV, acute lymphoblastic appearance leukemia, chronic lymphoid leukemia, Burkitt lymphoma, He Jiejin lymphomas, hairy cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, multiple myeloma, glioma, Walden Si Telun macroglobulinemia, cancer, melanoma, sarcoma, glioma, skin carcinoma, oral cancer, gastrointestinal cancer, colon cancer, gastric cancer, lung road cancer, pulmonary carcinoma, breast carcinoma, carcinoma of prostate, uterus carcinoma, endometrium cancer, cervical cancer, bladder cancer, cancer of pancreas, osteocarcinoma, hepatocarcinoma, carcinoma of gallbladder, renal carcinoma and carcinoma of testis.
45. the described method of claim 39, wherein said autoimmune disease are selected from nephritis, 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, 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, tabes dorsalis, giant cell arteritis/polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis or fibrosing alveolitis after acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus (sle), lupus nephritis, rheumatic fever, polyglandular syndrome, bullous pemphigoid, diabetes, purpura,Henoch-Schonlein, the streptococcal infection.
CN2010800386149A 2009-08-31 2010-08-30 Compositions and methods of use of immunotoxins comprising ranpirnase (RAP) show potent cytotoxic activity Pending CN102596235A (en)

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Application publication date: 20120718