大学精品课件：生物工程制药课件：lec10 Recombinant protein pharmaceuticals.ppt

1、Recombinant protein pharmaceuticalsn somatostatin(生长抑素)n Insulinn Hematopoietic Growth Factors(HGF)n Interleukins(IL)n Interferons(IFN)n Growth Hormones(GH)n Human Deoxyribonuclease(hDNase)n Tissue Plasminogen Activator(tPA)n Follicle-Stimulating Hormone(FSH)(卵泡刺激素)Expression in Escherichia coli ofa

2、 chemically synthesized genefor the hormone somatostatinn Itakura K,Hirose T,Crea R,Riggs AD,Heyneker HL,Bolivar F,Boyer HW.Science,1977 Dec;198(4321):1056-1063Somatostatin expression in E.colin A gene for somatostatin,a mammalian peptide(14 amino acid residues)hormone,was synthesized by chemical me

3、thods.n This gene was fused to the E.coli beta-galactosidaseSomatostatin expression in E.colin In vitro,active somatostatin was specifically cleaved from the large chimeric protein by treatment with cyanogen bromide(溴化氰).n This represents the first synthesis of a functional polypeptide product from

4、a gene of chemically origin.FDA-Approved Biopharmaceuticals(the first)nHumulin(recombinant human insulin)nEli Lilly&Co.nApproved for the treatment of diabetesExpression in Escherichia coliof chemically synthesizedgenes for human insulinnGoeddel DV,Kleid DG,Bolivar F,Heyneker HL,Yansura DG,Crea R,Hir

5、ose T,Kraszewski A,Itakura K,Riggs AD.Proc Natl Acad Sci U S A 1979 Jan;76(1):106-110Hematopoietic Growth Factors(HGF)Hematopoietic growth factorsRegulate hematopoietic celln proliferation:reproduction of similar cellsn differentiation:specialization or the acquisition of functions that are differen

6、t from the most primitive cell form(stem cell)n maturation:developmental changes that lead to functionally active cellsHematopoietic growth factorsInfluence virtually all organ systemsn hematopoiesis and immunocompetencen central nervous systemn cardiorespiratory systemn alimentary tract(消化道消化道)n li

7、vern bone remodelling(骨重建骨重建)n lipid metabolismn embryogenesis and maintenance of pregnancyHematopoietic growth factors the terminologynThe name of each hematopoietic growth factor is derived from its predominant target cell.n EPO=erythropoietinn G-CSF=granulocyte colony-stimulating factorn GM-CSF=g

8、ranulocyte macrophage colony stimulating factorn M-CSF=macrophage colony-stimulating factorHematopoietic growth factors Chemical propertiesn HGFs are glycoproteins.n HGFs have cysteine-cysteine disulfide bridges that dictate their three-dimensional configuration,which is necessary for biologic activ

9、ity.n Most HGFs are single-chain polypeptides weighing approximately 14 to 21 kD.n The carbohydrate content varies depending on the growth factor and production method,which in turn affects the molecular weight but not necessarily the biologic activity.How do hematopoieticgrowth factors function?n H

10、GFs act by binding to specific cell surface receptors.The resultant complex sends a signal to the cell to express genes,which in turn induce cellular proliferation,differentiation,or activation.n A HGF may also act indirectly if the cell express a gene that causes the production of a different HGF o

11、r another cytokine,which in turn binds to and stimulates a different cell.Human GM-CSF:nHuman GM-CSF:molecular cloning of the complementary DNA and purification of the natural and recombinant proteinsn Wong GG,Witek JS,Temple PA,Wilkens KM,Leary AC,Luxenberg DP,Jones SS,Brown EL,Kay RM,Orr EC,et al.

12、n Science 1985 May 17;228(4701):810-815Produce recombinant human GM-CSFin mammalian cellsn cDNA encoding the human GM-CSF were isolated by the construction of cDNA libraries in an expression vector and the screening of plasmid pools(60,000 bacterial colonies)by transient expression in monkey COS cel

13、ls.n Yield:recombinant GM-CSF:200 g/4 liters natural GM-CSF:4 g/40 litersFDA-Approved Biopharmaceuticalsn Leukine(GM-CSF)nImmunex Corp.nApproved for use in autologous bone marrow transplantation(自体骨髓移植)(March,1991).Commercially available rGM-CSFnProper name Trade namenMolgramostim LeucomaxnSargramos

14、tim LeukineIndications for rGM-CSFn1.Neutropenia(嗜中性白血球减少症)associated with myelosuppressive(骨髓抑制)cancer chemotherapyn2.bone marrow transplantationn3.antiviral therapy for AIDS-related cytomegalovirus(巨细胞病毒)n4.failed bone marrow transplantation or delayed engraftment(移植物植入)n5.mobilization of stem cel

15、ls for transplantationerythropoietin(EPO)nGlycoprotein that is produced in the kidney and stimulates red blood cell formation.Cloning and expression of thehuman erythropoietin genenLin FK,Suggs S,Lin CH,Browne JK,Smalling R,Egrie JC,Chen KK,Fox GM,Martin F,Stabinsky Z,et al.Proc Natl Acad Sci U S A,

16、1985 Nov;82(22):7580-7584Produce recombinant human EPO in mammaliancells(n PNAS USA 1985;82:7580-7584)n The partial amino-acid sequence of the purified EPO protein was determined(several peptides obtained by tryptic digestion)n The human erythropoietin gene has been isolated from a genomic phage lib

17、rary by using mixed 20-mer and 17-mer oligonucleotide probes.n The erythropoietin gene(4.8 kb),when introduced into Chinese hamster ovary(CHO)cells,produces erythropoietin that is biologically active in vitro and in vivo.FDA-Approved Biopharmaceuticalsn EPOnEPOGENnAmgen Ltd.nApproved for use in trea

18、ting anemia associated with chronic renal failuren(June,1989).Commercially available rhEPOnEpoetin alfa USA,China,JapannEpoetin beta Europe,JapanIndications for rhEPOn1.anemia associated with chronic renal failuren2.anemia of zidovudine-treated HIV-infected patientsn3.anemia of cancer chemotherapyn4

19、.reduction of allogeneic（异基因的）blood transfusions in surgery patients(hasten erythroid(红细胞)recovery in surgery patients)Cytokinesn Cytokines are soluble mediators or glycoproteins that aid in the communication between cells,primarily cells of the immunological,hematological,and neurological systems.n

20、 There are at least 60 different types of cytokines.e.g.interleukins,interferons,HGFsCytokines classificationn By their sourcenlymphokines lymphocytesnmonokines monocytesn By their functional definitionninterleukin “inter-leukocyte”,a cytokine which communicates between white blood cells.ninterferon

21、 a cytokine which interferes with the viral infection of a cell.Interleukins(IL)n A variety of glycoproteins produced by leucocytes(白细胞)and that regulate immunological responses.n Interleukins are often designated as IL-(number)and currently numbered up to IL-33.Interleukin-2(IL-2)n While many of th

22、e ILs are in clinical studies,only IL-2 and IL-21 are currently approved by the FDA and commercially available for clinical use.n IL-2 is synthesized and secreted primarily by T cells.n IL-2 was originally described as T cell growth factor(TCGF).n IL-2 can stimulate the growth,differentiation and ac

23、tivation of T cells,B cells,and NK cells.Structure and expression of a clonedcDNA for human interleukin-2nTaniguchi T,Matsui H,Fujita T,Takaoka C,Kashima N,Yoshimoto R,Hamuro J.Nature 1983 Mar 24-30;302(5906):305-310n A biologically active polypeptide,characteristic of human IL-2,was produced when t

24、he IL-2 cDNA was fused to a simian virus 40 promoter sequence and used to transfect cultured monkey COS cells.Biological activity of recombinant human interleukin-2 produced in Escherichia colin Rosenberg SA,Grimm EA,McGrogan M,Doyle M,Kawasaki E,Koths K,Mark DF.Science 1984 Mar 30;223(4643):1412-14

25、14n rIL-2 was not glycosylated.n rIL-2 was purified to apparent homogeneity and tested for biological activity in a variety of assays in vitro and in vivo.n No functional differences between native and the recombinant interleukin-2 molecules have been detected.Modulation of human natural killer cell

26、 activity by recombinant human interleukin 2nShaw AR,Bleackley RC,Merryweather JP,Barr PJ.Cell Immunol 1985 Feb;90(2):547-554n Recombinant human IL-2,secreted by yeast harboring a plasmid containing a synthetic IL-2 gene,is biologically active in augmenting human natural killer(NK,自然杀伤细胞)cell activi

27、ty.FDA-ApprovedBiopharmaceuticals rIL-2n Proleukin(aldesleukin)nChiron Corp.n Approved for1.the treatment of kidney carcinoma(May,1992).2.treatment of metastatic melanoma(Jan.1998)(转移性黑素瘤).nlec10 Recombinant protein pharmaceuticals-continuednlec11 Monoclonal antibodynlec12 vaccineInterferons(IFNs)nA

28、 family of glycoproteins produced in mammals that interfered with viral replication in cells.Synthesis in E.coli of a polypeptide withhuman leukocyte interferon activitynNagata S,Taira H,Hall A,et al.,Nature 1980 Mar 27;284(5754):316-320.n ds cDNA prepared from the 12S fraction of poly(A)RNA from in

29、terferon(IF)-producing human leukocytes was cloned in E.coli using the pBR322 vector.n One of the resulting clones had a 910-base pair insert which could hybridise to IF mRNA and was responsible for the production of a polypeptide with biological IF activity.Human leukocyte interferon produced byE.c

30、oli is biologically activen Goeddel DV,Yelverton E,Ullrich A,et al.,Nature 1980 Oct 2;287(5781):411-416n A human leukocyte interferon cDNA was enzymatically synthesized,inserted into the vector pBR322,and cloned in E.coli(cDNA codes for a 23-amino acid signal peptide followed by an interferon polype

31、ptide of 165 amino acids).n An expression plasmid was constructed which permits the synthesis in E.coli of 2.5 108 units of interferon per litre of culture.n This LeIF protected squirrel monkeys(松鼠猴)from lethal encephalomyocarditis（脑心肌炎病毒）virus infection.Expression of a human gene forinterferon in y

32、eastn Hitzeman RA,Hagie FE,Levine HL,et al.,Nature 1981 Oct 29;293(5835):717-722n A DNA sequence coding for mature human leukocyte interferon D(LeIF-D)was linked with DNA fragments of the 5-flanking sequences of the Saccharomyces cerevisiae(yeast)alcohol dehydrogenase I gene in a plasmid capable of

33、autonomous replication and selection in both yeast and E.coli.n Yeast cells transformed by these plasmids synthesize up to 1 x 106 molecules of biologically active LeIF-D per cell.FDA-ApprovedBiopharmaceuticals IFN-n Intron A(recombinant interferon-2a)nSchering-Plough Corp.n Approved for the treatme

34、nt ofnhairy cell leukemia(June,1986)ngenital warts（尖锐湿疣）(June,1988)nAIDS-related Kaposis sarcoma(卡波西肉瘤)(November,1988)nnon-A,non-B hepatitis(February,1991)nhepatitis B(July,1992).FDA-ApprovedBiopharmaceuticals IFN-n Roferon-A (recombinant interferon-2b)nHoffman-La Rochen Approved for the treatment o

35、fnhairy cell leukemia(June,1986)nAIDS-related Kaposis sarcoma(Nov.,1988)nchronic myelogenous leukemia（慢性骨髓性白血病）(Oct.,1995)nhepatitis C(Nov.,1995)Expression of human fibroblastinterferon gene in Escherichia colin Derynck R,Remaut E,Saman E,et al.,Nature 1980 Sep 18;287(5779):193-197.n A thermoinducib

36、le expression plasmid under control of the phage lambda PL promoter.n Hybrid proteins:starting with beta-lactamase or phage MS2 polymerase information followed by the total preinterferon.n On induction,antiviral activity,whose physicochemical,immunological and biological characteristics closely corr

37、esponded to those of authentic human fibroblast interferon,was synthesized.Expression of the human fibroblastinterferon gene in Escherichia colinTaniguchi T,Guarente L,Roberts TM,et al.,Proc Natl Acad Sci U S A 1980 Sep;77(9):5230-5233n Two plasmids were constructed to direct expression in E.coli of

38、 the human fibroblast interferon(F-IF)gene.pre-F-IF and mature F-IFn Extracts of bacteria synthesizing mature F-IF display antiviral activity characteristic of human F-IF.No such activity was detected in extracts of bacteria synthesizing pre-F-IF.FDA-ApprovedBiopharmaceuticals IFN-n Betaseron(recomb

39、inant interferon-1b)nBerlex Laboratories/Chiron Corp.n Approved for use in the management of relapsing-remitting multiple sclerosis（复发-缓解型多发性硬化症）(August,1993).Expression of human immune interferoncDNA in E.coli and monkey cellsnGray PW,Leung DW,Pennica D,et al.,Nature 1982 Feb 11;295(5849):503-508.M

40、olecular cloning of human immuneinterferon cDNA and its expressionin eukaryotic cellsnDevos R,Cheroutre H,Taya Y,et al.,Nucleic Acids Res 1982 Apr 24;10(8):2487-2501n monkey cellsFDA-ApprovedBiopharmaceuticals IFN-n Actimmune(recombinant interferon-1b)nGenentech,Inc.n Approved for use in the managem

41、ent of chronic granulomatous disease（慢性肉芽肿病）(December,1990).Human Growth Hormones(hGH)n also known as somatotrophin（促生长素;生长激素）n two hGH genes:hGH-N:“normal”,major circulating form in pituitary gland hGH-V:“variant”form in placentan essential for normal growth and development in humanHuman growth hor

42、mone:complementaryDNA cloning and expression in bacterian Martial JA,Hallewell RA,Baxter JD,et al.,Science 1979 Aug 10;205(4406):602-607n hGH cDNA contains 29 bp 5 UTR,651 bp coding for the prehormone,and 108 bp 3 UTR.n hGH cDNA has been linked in phase to a fragment of the trp D gene of E.coli in a

43、 plasmid.n A fusion protein is synthesized at high level(ca 3%of bacterial protein)under the control of the regulatory region of the trp operon.n This fusion protein reacts specifically with antibodies to hGH and is stable in E.coli.Direct expression in Escherichia coli of a DNA sequence coding for

44、human growth hormonenGoeddel DV,Heyneker HL,Hozumi T,et al.,Nature 1979 Oct 18;281(5732):544-548FDA-ApprovedBiopharmaceuticals hGHn Protropin(met-rhGH produced in E.coli)nGenentech,Inc.n Approved for treating human growth hormone deficiency in children(May,1985).n Met-rhGH is more effective than phe

45、-hGH.However,immunogenicity is a problem.FDA-ApprovedBiopharmaceuticals hGHn Humatrope(rhGH produced in E.coli)nEli Lilly&Co.n Approved for treating human growth hormone deficiency in children(March,1987).FDA-ApprovedBiopharmaceuticals hGHn Saizen(rhGH produced in mammalian cells)nSerono,Inc.n Appro

46、ved for treating human growth hormone deficiency in children(Oct.1996)Human Deoxyribonuclease(hDNase)n hDNase normally is present in saliva,urine,pancreatic secretions and blood.n hDNase catalyzes the hydrolysis of extracellular DNA into oligonucleotides.Cystic fibrosis(CF,囊性纤维囊性纤维化病化病)na hereditary

47、 disease starting in infancy that affects various glands and results in secretion of thick mucus that blocks internal passages,including those of the lungs,causing respiratory infections.nMost CF patients died before the age of thirty.Cystic fibrosis(CF)n viscous mucus:obstruction of the airways and

48、 pancreatic ductsn viscous:mucus glycoproteins and DNAn high concentration(3-14 mg/ml)DNA make the secretions very viscous bind aminoglycoside antibiotics commonly used for treatment of pulmonary infections(eg.hygromycin B,kanamycin,streptomycin)Recombinant Human DNase I Reducesthe Viscosity of Cyst

49、ic Fibrosis Sputum（唾液唾液 痰痰）nShak S,Capon DJ,Hellmiss R,Marsters SA,and Baker CL,PNAS 1990 87:9188-9192FDA-ApprovedBiopharmaceuticals rhDNasen Pulmozyme (dornase alpha)nGenentech,Inc.n Approved for use in the management of cystic fibrosis(December,1993)PTC124 targets genetic disorders caused by nonse

50、nse mutations n Nature 447,87-91(3 May 2007)nNonsense mutations promote premature translational termination and cause anywhere from 570%of the individual cases of most inherited diseases.Studies on nonsense-mediated cystic fibrosis have indicated that boosting specific protein synthesis from 1%to as