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Molecular Biotechnology

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Molecular Biotechnology

  1. 1. ~: ,., ‘ "'13" 2 _, ,.. ._ ,5 . ,._ ‘EL’ 5'5, «-. .a ,3. at 310’: echnology _ “A . .. . T 1./ Lole c 1 Ther ape ut ic Agent s Ch u ‘.1 n —1t-1 u Ch c n , P 11 . D. % Associate Professor Department of Life Science and Institute of Biomedicine National Chung Hsine Universitv
  2. 2. w. o.. J.av.4HM 4 0.. .. :u; . ; .. ..C? ... ... it mo. aifiuaass ~/ hwicto V O . Q Q . B. 44 0 909 . . w. ... £a1Z. ... »J. ,.. UL mz. . . o . . . 1. ? .»1*. ._. ... .u -. ... .e. .xw. .. . .s . . x. . . .. .3. I . . L fa e 4 ». rr . .. mo . . . ..q. . . . . . 73.. ... . M. w to J _ . .. . 3 . - w_/ A . p / I _ . I la . u Was . l_ Iv . 9 W0. - . . .1? . ..H a . 1 . . s so». s .7._; M . .. |.. _i. .. . .. _ . &r st)». .. |l « 2 5 , . .1 mil . s.F 5 v we _. ... o4a”. . :1 _ + a1, . .rJ_. . In. a. r. .,. ... .._. ,3.1 .3. .1 v $1.‘. . 1.4! . T I>. I I L 3L. . ... m.; -:: + . . is It‘. .. I.. §l -3» A . ofi 1|. Ls'1ta. ..iI. .. . ... .+. ... an . .0. 3 4. 7.. ... . ,3 "L. S . . . .+ . . . .. u . . J 9,. It I I r J 'Ul’. ’.. 4_II nix » V xx .1. X1 in T14 .3. . .1I| a.-«J. » —
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  4. 4. Monoclonal antibodies as ‘ ‘ therapeutic agents: Background: 1. Hundred years ago. horses were inoculated with C. diphtheriae. The crude hors antiserum was used to treat a fatal childhood diphtheria disease. 2. This kind of antibody therapy carries considerable risks and is not widely used today. Patients often develop their own anti- bodies against the foreign proteins. 3. Using hybridoma technology. pure monoclonal antibodies are once again seen as potential therapeutic agents.
  5. 5. (I) Structure and function of ant ibodie s 5‘ Structure of antibody molecule: Antigen binding 1. Two identical light chain (L): / activity domain 2. Two identical heavy chain (H): is ' 3. H & 1. chains held together » iT'i~: 't r . by 11- bonding and disullidc t/ /t-<1;’~iL‘)p; ~r, ‘- " linkage: T if i S ' . , W 4. CDRs regions for recognize . ;,. n: and bind antigens that lie . __ g p _ within variable ( V“ &V, ) r’ M r_ g ‘ _-__{__, ,,’ 7»; ._ regions in N- terminal ends; a _*. _,+" 1.. A , _ 5. One constant domain (CL) in L ' 9 I A chain, and three constant domains y ' - * . if p (C, ,,__, ) in H chain; “ 6. Two Fab and one Fc are presented after papain protcolytic digestion. “ l l = rtwzi «‘t‘>ii, Figure 10.1.?
  6. 6. The act ion of antibodies it In an intact antibody molecule, the Fc portion elicits several immunological responses after ant igen- antibody binding occurs: ‘it I . The complement cascade is activated. The complements of this system break down cell membranes, activate phagocytes, and generate signals to immune response. 2. Antibody- dependent cell- mediated cytotoxicity (AADCC) is produced, which is the result of Fe binding to an Fc receptor of an ADCC effector cell. 3. After the Fab region binds to a soluble antigen, the Fc portion of an antibody can be bound to Fc receptors of phagocytic cells, which engulf and destroy the ant ibody- antigen complex.
  7. 7. .In the 1970s, passive immunization was reconsidered (ll) Preventing rejection of transplanted organs as a way of preventing immunological rejection of a transplanted organ. . The mouse mAb OKT3 was the first to be approved by the US FDA for use as an immunosuppressive agent after organ transplantation in human. . The OKT3 mAb binds to a cell surface receptor called CD3, which is present in all T cells, to prevent a full immunological response and reduce organ rejection.
  8. 8. (I I I) Chemically linked monoclonal ant ibodie s *5‘ A number of different strategies may be used to enhance the delivery of a drug to its target site: 1. Drugs can be encapsulated in liposomes 2. Certain toxin genes‘ may be incorporate into tumor- infiltrating lynipliocytes; 3. Some drugs can be coupled to in; —b tha are specific for proteins found only on the surface of target cells; 4. may be used. The specific mAb COLlpllI enzyme can trigger activity of prodrug Figure 10.14 A Inert forms of some drugs, called proc ~' ‘l E! —xCeé? l surface protein "j— Target tell I/ I Kg/ l / .~'‘‘’ f —. »l0norlonai ant: l>r. J:l' V — IiirgL'i tell
  9. 9. An example for antibody " therapeutic agent in thrombus * The majority of the natural deaths in North American & Europe are the cerebral and coronary artery by a blood clot t ing (T h r omb us ). *5‘ The mechanism of plasmin in degradation of blood fibrinogcn. Figure 10.15 Activation of plas- minogen to plasmin and break- ‘down of the two substrates (fib- rinogcn and fibrin) by plasmin in the blood system. Plasminogen activator Plasminogen —--—-5' Plasmin 7 Degradation products ‘ Degradation products
  10. 10. An example for antibody therapeutic agent in thrombus The designation of an immunot he rape ut ic t h r ombolyt ic agent. The antifibrin 1nAb is specific for the fibrin bound in blood clots, and is coupled to plasminoge n activator (PA). Figure 10.16 Structure of an immuno- therapeutic thrombolytic agent. Anti- fibrin antibody, a monoclonal antibody that is specific for the fibrin found in blood clots, is coupled to plasminogen activator (PA). After the complex binds to the fibrin of a blood dot, the plas- minogen activator causes plasmin to accumulate in the vicinity of the clot. The plasmin then degrades the clot. Plasminogen ? —-F- Plasmin
  11. 11. (VI) Hybrid human- mouse monoclonal ant ib odie s
  12. 12. U€I1€l ically €I1g1I1€€I'€(1 humanized antibody W n a to. u M n . m 4.. Mn 0 n u m . .m L . d n a 4». F. e n E ab 9. an. m 0 . n N R D C m, .l . w lonal . intibod_' r('pl. cc the CDRs of the genes for a human antibody. The act of this constructed gene IS an immunoglobulin with the antigcri binding nantibody molecule (light blue) (URI CDR. ‘
  13. 13. The approaches for engineered humanized antibody Figure 10.19 PCR amplification of CDR1 from a rodent monoclonal antibody L chain CDNA. The PCR primers P1 and P2 contain oligonucleotides complementary to the rodent CDR1 DNA. In addition, P1 and P2 each contain 12 nucleotides at their 5 ’ end that is complementary to the framework regions of human monoclonal L chain cDNAs. Using six separate pairs of oiigonucleotide pr-imcrs—three for the V. , region and three for the VH region—each of the rodent CDRs is separately amplified by PCR. Then, by oligonucleotide-directed mutagenesis, the amplified rodent CDRs are spliced into human antibody genes in place of the resident CDRs. This grafting is made possible by the presence of DNA complementary to the human framework regions on the amplified rodent CDR DNAs. Signal ‘ peptide PR1 CDR1 FR2 CDR2 PR3 CDR3 FR4 — / / / / ‘x / / 4! / P1 CDRI ‘t>_2
  14. 14. V1. Production or antibodies in E. coli Hybridoma cells, grow relatively slowly, do not attain high cell density, and require a complex co»--«~«'~~-mm and expensive growth medium. L-(m', ~«_; *-_-_‘ < ¢_, ..i. ,.(. m.. r. Bacteria to act as “bioreactor" for the production of mAb has ‘ Cunlh-rs: N am‘! ( rluarn a n xpv bu lump 0- --u tr been obtained. ficnun punt. “ Ia: Amiga bmamg The procedures to create Ecoli _ ; combinatorial library for VL & VH. lmnfluom 1 LED‘! -Mn pl. nwnad—lLl. rhino [INK cnnslnxv n1-11 arvgrn h-min: . Imam: -rt turn I (on
  15. 15. use or lVll.5 pnage to produce a combinatorial library synthesized Fv antibody fragments H chain ( DNA I. z_h. nn c DNA LPCR lricn V” Linker VL I. Z _ J lngaln Linker VL I: :Y” is Splu v inlu Imclc-m: »ph.1g9 M13 vector into Ml3 gcnc V“ Linker / L Pm’ . ,0 n1ndifi(~d N11 _ NA mrn M1 ‘1 l. -. , I) I L)! ‘ l <
  16. 16. Disulfide— stabilized Fv immunotoxin is more effective than single- chain Fv molecule ‘ii A B ‘I/ xxx‘ Kfi r: l l i : l W -' Vt VH "“’d L Figure 10,25 Schematic repre- l Kaoxm scntanon or a single-chain Fv im- ' ‘. ,/’ munotoxin (A) and a disulfide— l_lFr(€F peptide Disulfidc bridge stabilized Fv immunotoxin (3).
  17. 17. HIV therapeutic agent s ‘ AIDS is caused by human immuno- < I‘ <5 W m Jay x . . Gina/ (NV s 095.0 a. a e aha ,0 - mm a , as o .0 D x 9 m g . . _ _. . , Z s. ‘ , TH i. .. U H 0 p o _ ,1. 0 - 0.. ed W 3 1 deficiency virus (HIV), so far still unable to produce effective vaccine. ‘ The target cell for HIV is destroyed the TH lymphocyte of the immune system by an HIV glycoprotein (gpl20) attached to the TH cell receptor, called CD4 . ‘ A CD4- Fe immunoadhesin has been developed to destroy HIV virus. §“
  18. 18. Genetic construction of a CD4 — Pse ua’0122011as e xot oxin For specific killing lll“'— infected L‘L‘ll ’P[aSr. nid Binds to HIV- Leads the exotoxin Inactivates the Infected cell fusion protein enter EF- 3 and killed surface to the infected cell the iI1f€CI€C| Cell : ' E "T - x 7 7 H x p f or immunot he r a py . ~ T7 ‘= CD4 Exotoxin Exotoxin ‘ promoter domain domain II domain III Ribosomal binding ' %—$ site
  19. 19. {if V. Nucleic Acids as therapeutic agents l i
  20. 20. V. Nucleic Acids as therapeutic agents 2. Antisense Oligonucleotide Technology
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  22. 22. 7% V. Nucleic Acids as therapeutic agents ’ “ Mn-‘M 1 5, l""‘“"""'"‘"‘ i----- %. ... ... ..
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