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  • 1. Antibody generation T-cells: cell mediated immune reactions and B-cells:  secreted antibodies 2002 Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter. 23-1
  • 2. Prexisting B cells that are already producing antibodies that can bind to a specific antigen are stimulated to divide when presented with that antigen. There are many different clones of such precursor cells, each of which is stimualted. The final response is therefore POLYclonal. 2002 Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter. 23-2
  • 3. The antbody secreting effector cells terminalyl differentiate (die) but their sister memory cells live on to generated an amplified reponse upon a second exposure to antigen 2002 Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter. 23-3
  • 4. 2002 Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter. 23-4
  • 5. Monoclonal antibodies via cell hybridization Selects for rare hybrid cells Spleen cells do not grow in culture TGr myeloma cells do not grow in HAT e.g., in peritoneal cavity) 23-5
  • 6. Monoclonal antibody generation Hprt- myeloma cells 6-TG-resisatnt HAT- (HAT) Plate among many wells for supernantent testing Plating positives at low density (~1/well) for cloning 23-6
  • 7. Reduced S-S bonds Unreduced Myeloma hybrid myeloma hybrid Kohler, G., and C. Milstein (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495-497. Fused a myelolma cell line to spleen cells from s amouse immunized against sheep red blood cells. Established cell lines (hybridomas) that secrete any antibody that can be raised in a mouse. Use of myeloma parent obviated extinction (shut-off) of Ig genes by the immortal parent. Cesar Milstein Isoelectric focusing (not SDS PAGE) (MRC = Medical Research Council) Isoelectric focusing of immunoglobulins made in the hybridoma cells  new bands Complexity not explained; perhaps not truly clonal.. Later used a myeloma mutant parent that no longer secreted an endogenous immunoglobulin. Georges Kohler 23-7
  • 8. MAb therapy targets Inflammation Autoimmune disease Graft rejection Cancer Viral infection 23-8
  • 9. Therapeutic strategies Plain MAbs MAbs fused to other protein binders (e.g., soluble receptors) to increase avidity and/or to effect ADCC MAbs fused to cytotoxic agents (toxins, radionuclides) Toxins: ricin (stops protein synthesis) calicheamicin (DNA breaks) Radionuclides: 90Y = yttrium 111I = indium 23-9
  • 10. Sme MAbs approved for human therapy Sponsor company Generic name US trade mAb type Therapeutic US name category approval Orthoclone Johnson & Johnson Muromonab-CD3 Murine Immunological 19.06.1986 Transplantation OKT3 Centocor Abciximab ReoPro Chimeric Hemostasis 22.12.1994 Stroke Biogen IDEC Rituximab Rituxan Chimeric Antineoplastic 26.11.1997 Lymphoma Protein Design Daclizumab Zenapax Humanized Immunological 10.12.1997 IL-2, immunosuppressant Labs | Novartis Basiliximab Simulect Chimeric Immunological 12.05.1998 Transplantation Respiratory infection MedImmune Palivizumab Synagis Humanized Anti-infective 19.06.1998 Synciitial Virus Centocor Infliximab Remicade Chimeric Immunological 24.08.1998 Arthritis Genentech Trastuzumab Herceptin Humanized Antineoplastic 25.09.1998 HER-2/neu (EGF2) breast cancer Gemtuzumab Wyeth Mylotarg Humanized Antineoplastic 17.05.2000 CD33 leukemia (AML) ozogamicin Millennium/IL Alemtuzumab Campath Humanized Antineoplastic 07.05.2001 Leukemia EX Ibritumomab Biogen IDEC Zevalin Murine Antineoplastic 19.02.2002 Lymphoma tiuxetan Abbott Adalimumab Humira Human Immunological 31.12.2002 Arthritis Genentech Omalizumab Xolair Humanized Immunological 20.06.2003 IgE asthma Corixa Tositumomab-I131 BEXXAR Murine Antineoplastic 27.06.2003 Lymphoma Genentech Efalizumab Raptiva Humanized Immunological 27.10.2003 Psoriasis Imclone Cetuximab Erbitux Chimeric Antineoplastic 12.02.2004 EGF-R colon cancer Systems Genentech Bevacizumab Avastin Humanized Antineoplastic 26.02.2004 VEGF colon cancer 23-10
  • 11. Monoclonal antibody generation - Cells needed myeloma cells and mouse spleen cells - antigen administration Kohler and Milstein - hybridoma formation via cell fusion - selection mutants required (myeloma hprt- usually) - antibody generation cDNA cloning - engineered MAbs expression vectors - refinement chimeric, humanized human, truly human 23-11
  • 12. Problems of mouse MAbs 1) Fc portion limited in its ability to interact with Fc receptors of human cells. 2) Lower serum half-life 3) Development of human anti-mouse antibodies (HAMA) A) Retreatment results in allergy or anaphylactic shock Breedveld, Lancet 2000 355:9205 B) Retreatment is less effective Solutions via recombinant DNA genetic engineering : 1) Chimeric mouse-human antibodies: mouse V regions fused to Hu C-region 2) Humanized mouse antibodies, Parts of V-region from human interspersed with mouse CDR V-regions 3) Human antibodies (fully), via transgenic mice carrying human immunoglobulin genes (Medarex, Abgenix, Kirin) CDR = complementarity-determining region 23-12
  • 13. MAb Fusion Proteins Other protein-binding proteins: natural receptors in soluble form Analogous to MAbs and make use of the Fc portion of the antibody molecule: Example: Enbrel (etanercept): Anti-rheumatoid arthritis drug Soluble TNF receptor fused to the Fc IgG1 domain (TNF= tumor necrosis factor) Ties up TNF, blocking its inflammatory function Fc domain dimerizes the receptor, which increases its affinity for TNF. Fc domain increases the half-life of the protein in the bloodstream Amgen + Wyeth Still experimental: Anti-HIV drug PRO 542 Uses soluble form of the CD4, the molecule to which HIV attaches on T-cells Soluble CD4 (HIV receptor) fused to IgG2. Tetrameric (all 4 V-regions replaced) – therefore mutlivalent Reduced Fc function (chose IgG2 for this reason) Better half-life than soluble CD4 itself (Recently replaced by a MAb (PRO 140) targeting the CCR5 cell surface protein, required for viral entry) Progenics TNF = tumor necrosis factor) 23-13
  • 14. Single chain antibodies (scFv) Ag binding site 15 AA linker 23-14
  • 15. Phage display to isolate functional V-regions Phage display can be used to screen billions of V-region variants for binding to a particular antigen of choice Key requirement of this powerful strategy, and many like it: A physical link of 1) a nucleic acid sequence (here, DNA) to 2) the phenotype (e.g., binding to something) of a protein coded by that nucleic acid the protein the DNA (inside) “Panning” 23-15
  • 16. Phage display selection of scFv’s (single-chain F-variable regions) Source of sequence: PCR from genome or RT-PCR from mRNA, add randomization (doped synthesis). or 23-16
  • 17. Two types of protein glycosylation N-acetyl group glucose galactose 23-17
  • 18. = Penta- saccharide common = core All shown Triantennary Diantennary here, (also tetra-antennary) With bisecting GlcNAc N-linked With fucosylated core (to amide N of Asn in N-X-S Substantial in size or N-X-T) Carbohydrates attached to loops Fucose or near termini Also O-linked, to ser or thr (hydroxyl on side chain) 23-18
  • 19. Figure 7.28. Examples of O-linked oligosaccharides O-linked oligosaccharides usually consist of only a few carbohydrate residues, which are added one sugar at a time. 23-19
  • 20. Carbohydrate structure specific for: Cell type Physiological state No. of sites depends on 3-D structure of protein Structure at that site depends on the site E.g., transferrin from different cell types : Cerebrospinal fluid (made in brain): diantennary asialo agalacto fucosylated bisecting GlcNAc Blood (made in liver): diantennary NAcNeu (sialated= sialic acid) Sialic acid structure: see next graphic afucosylated 23-20
  • 21. neuraminic acid – one of the sialic acids = : both terms are used, confusedly NAcNeu: Glycerol moiety Carboxyl (acid) Mannose framework Acetylated amino group deoxy 23-21
  • 22. Glycosylation pattern affects signaling, for: Delivery of the glyco protein to the right cell receptor for activity Its clearance rate Microheterogeneity: Lots of isoforms typically present Glycosylation does not seem to represent a bottleneck in high-producing cells: 0.1 mg/l  (amplify)  200 mg/l = same pattern Insect cells (Baculovirus, high level transient expression): Too simple a pattern compared to human Mouse and hamster cells: similar to human Hamster: less heterogeneity 23-22
  • 23. Genetic engineering of glycosylation to: Modify or enhance activity E.g.: Better binding to a receptor More specific binding Different binding Also: Antigenicity Clearance rate Decrease microheterogeneity (for clinical application) 23-23
  • 24. Got this far 23-24
  • 25. Modifying glycosylation 1. Add or subtract sites to your favorite protein (cis) 1a. Subtract sites: Easy, change N or S or T to A by site-directed mutagenesis 1b. Add sites: Not so easy. Consensus N-X-S does not work, e.g.: requires the insertion of a ~12 aa region encompassing a real N-glycosylation site (6 suffices for O-linked) Place on an end or on a loop (must know protein’s structure) Works 2. Change the general glycosylation phenotype of the host cell (trans) E.g., Pam Stanley: lectin-resistant mutants 23-25
  • 26. Modifying glycosylation 1. Add or subtract sites to your favorite protein (cis) 2. Change the general glycosylation phenotype of the host cell (trans) 2. Clone enzyme genes: Glycosyl transferases, mostly Also some synthetases (e.g., NAcNeu) Can be complex: e.g., 7 different fucosyl transferases (FTs), with different (overlapping) substrate specificities Simpler example: Hamster cells do only 2,3 sialylation. Humans do 2,6 as well, via a 2,6 sialyl transferase (ST) Experiment: Over-express cloned human 2,6 ST, along with a substrate protein. producing permanent transfectants of BHK cells (BHK = baby hamster kidney) Works: Get both types of structures now, substantially (although not exactly the same ratio as in human cells). J Biol Chem, Vol. 273, Issue 47, 30985-30994, November 20, 1998 In Vivo Specificity of Human 1,3/4-Fucosyltransferases III-VII in the Biosynthesis of LewisX and Sialyl LewisX Motifs on Complex-type N-Glycans. COEXPRESSION STUDIES FROM BHK-21 CELLS TOGETHER WITH HUMAN -TRACE PROTEIN Eckart Grabenhorst , Manfred Nimtz , Júlia Costa§, and Harald S. Conradt ¶ 23-26
  • 27. Isolate mutant mammalian cell lines deficient in specific glycosylation enzymes Stanley: Isolation of multiply mutated glycosylation mutants by selecting for lectin resistance Lectins = carbohydrate-binding proteins Plant lectins used mostly here (but occur widely in animals as well) Sequential selections, push - pull on resistance, sensitivity Resistance: enzyme deficiency  failure to add the sugar need for lectin binding Increased sensitivity: failure to add a sugar produces greater exposure of underlying sugars in a transferase-negative mutant  better binding to the exposed sugar Showed power of selection Showed usefulness of complementation analysis via cell hybridization Pam Stanley Hybrid selection: All lec-R mutants were: WGA (wheat germ agglutinin) resistant (various degrees) & pro- Tester parent was single lec-R + GAT- (req. glycine, adenine and thymidine) Select in medium lacking pro, GAT, and with +/- WGA Complementing hybrids will have regained sensitivity to WGA Mutants in the same gene will remain WGA resistant (non-complementation) Could now be used as a tabla rasa (blank slate) introducing a series of enzymes to build custom tailored glyco-conjugates. Complicated though (order of addition, location in the Golgi, etc. ) Potential: targeting to carbohydrate-sensitive receptors (e.g., liver asialoglycoprotein receptor); clearance rate Review: Nature Biotechnology 19, 913 - 917 (2001) , The bittersweet promise of glycobiology. Alan Dove 23-27
  • 28. Umana, P., Jean-Mairet, J., Moudry, R., Amstutz, H., and Bailey, J.E. 1999. Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nat Biotechnol 17: 176-180. Target here (bisecting NAcG) (NAcG = N-acetyl-glucosamine here) Presence of the bisecting NAcG enhances binding of T-cell receptor to the Fc region of antibodies. Binding is needed for ADCC. Mouse and hamster cell lines used for commercial production lack the glycosyltransferase needed for bisecting NAcG addition A rat myeloma cell line does produce MAb with the bisecting NAcG. Hypothesis: Expression of the rat enzyme in a CHO cell line will add a bisecting NacG to the anti-neuroblastoma MAb produced by these cells. The modified MAb will be a better mediator of ADCC. Experiment: Clone the cDNA for this enzyme from the rat line and transfer it to CHO cells, driven by an inducible tet promoter. Check sugar structure of MAb and ADCC efficiency of the MAb. 23-28
  • 29. TARGET CELL (Killer T-cell) Genentech Commercial MAb injected as a therapeutic T-cell surface receptor binds Fc region of antibody molecule 23-29 (Fc gammaR)
  • 30. Protein Glycosylation Assigned: Naoko Yamane-Ohnuki, et al.. Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity. Biotechnol Bioeng. 2004 Sep 5;87(5):614-22 Optional Update: Kanda Y, Yamane-Ohnuki N, Sakai N, Yamano K, Nakano R, Inoue M, Misaka H, Iida S, Wakitani M, Konno Y, Yano K, Shitara K, Hosoi S, Satoh M. Comparison of cell lines for stable production of fucose- negative antibodies with enhanced ADCC. Biotechnol Bioeng. 2006 Jul 5;94(4):680-8. Umana, P., Jean-Mairet, J., Moudry, R., Amstutz, H., and Bailey, J.E. 1999. Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nat Biotechnol 17: 176-180. Review: Grabenhorst, E., Schlenke, P., Pohl,., Nimtz, M., and Conradt, H.S. 1999. Genetic engineering of recombinant glycoproteins and the glycosylation pathway in mammalian host cells. Glycoconj J 16: 81-97. Background: Stanley, P. 1989. Chinese hamster ovary cell mutants with multiple glycosylation defects for production of glycoproteins with minimal carbohydrate heterogeneity. Mol Cell Biol 9: 377-383. 23-30
  • 31. Biotechnol Bioeng. 2004 Sep 5;87(5):614-22 Hypothesis: Fucose interferes with binding of the T-cell Fcgamma3 receptor to the Fc region of an antibody molecule. Elimination of fucose from produced MAbs will increase ADCC Create a mutant CHO cells (starting with amplifiable dhfr- cells) in which the fucose trasnferase genes have been knocked out. All MAbs produced in these mutant cells will be better at promoting ADCC 23-31
  • 32. Double knock-out strategy for FUT8 an alpha-1,6,fucosyl transferase Little sequence data available for Chinese hamster Isolate CHO cDNA using mouse sequence data for primers Use CHO cDNA to isolate CHO genomic fragments from a commercial lambda library K.O. exon 1 translation start region Homology regions DT= diphtheria toxin gene, Kills if integrated via For hemizygote: non-homologous recombination Select for G418 resistance, Screen by PCR for homologous recomb. 108 cells  45000 colonies 40 false recombinants (extension-duplications) + 1 true recombinant Step 2 for homozygote, select for Pur-resistance 1.6X10870,000 screened  Lox sites 10 double KO homozygotes. Remove drug resis. genes by transient transfection with Cre Recombinase. Exon 1 suffers a 200 nt deletion Note: 10’s of thousands of PCRs performed to screen for homologous recomb., using 96-well plates 23-32
  • 33. Double knockout evidence After Cre treatment Original KO’d genes have a 1.5 kb insertion (Southern blot) mRNA has 200 nt deletion (RT-PCR 23-33
  • 34. Use of a fluoresceinated lentil lectin (LCA) that binds fucose oligosaccharides to demonstrate lack of fucosylation in glycosylated proteins in the FUT8 -/- cells Control background fluorescence (FL-anti avidin) FUT8 +/+ FUT8 +/- Surprising: CHO cells do not have excess fucosylation capacity . . . FUT8 -/- 23-34
  • 35. Rituxan (anti-CD20) produced in FUT -/- cells does not contain fucose (HPLC analysis) Digestion all the way to monosaccharides Missing d - g 23-35
  • 36. Binding to CD20 membranes In ADCC, FUT8-/- anti-CD20 >> Rituxan FUT8-/- anti CD20 = Ritxuan Anti-CD20 from a partially FUT-deficient rat cell line Fc-Receptor protein binding assay Rat line FUT-/-’s Complement-mediated cell toxicity is the same for FUT8-/- and Rituxan Rituxan = commercial product, 98% fucosylated 23-36
  • 37. Very laborious, but apparently a big payoff. Better selection?: Why not use the fluorescent LCA to select for the FUT8 KO’s along with G418 resistance (double sequential selection)? 23-37

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