Antibodies can participate in host defense in three main ways

1. MAbs continued Antibodies can participate in host defense in three main ways

2. ADCC = antibody-dependent cell-mediated cytotoxicity NK = Natural killer cells (T-cell class) FcgammaRIII = Fc receptor on NK cell surface Bound NK cells release granules with a protein that forms pores on the target cell (perforin) and an enzyme that penetrates the target cell and induces apoptosis through a caspase (protease) cascade.

3. TARGET CELL (Killer T-cell) Genentech Commercial MAb injected as a therapeutic T-cell surface receptor binds Fc region of antibody molecule(Fc gammaR)

4. MAb therapy targets Inflammation Autoimmune disease Graft rejection Heart disease (thrombosis) Cancer Viral infection

5. Therapeutic strategies Mabs straight Mabs fused to other protein binders (e.g., soluble receptors) Mabs fused to cytotoxic agents (toxins, radionuclides) Toxins: ricin (stops protein synthesis) calicheamicin (DNA breaks) Radionuclides: 90Y = yttrium 111I = indium

6. Problems of mouse MAbs • Fc portion limited in its ability to interact with Fc receptors of human cells. • Lower serum half-life • Development of human anti-mouse antibodies (HAMA) • Retreatment results in allergy or anaphylactic shock • Retreatment is less effective Breedveld, Lancet 2000 355:9205 • Solutions via recombinant DNA genetic engineering : • Chimeric mouse-human antibodies: Hu V-region fused to mouse C regions • Humanized mouse antibodies, Parts of V-region from human interspersed with mouse CDR V-regions • Human antibodies (fully), via transgenic mice carrying human immunoglobulin genes(Medarex, Abgenix, Kirin) CDR = complemetarity-determining region

7. MAbs approved for human therapy Transplantation Stroke Lymphoma IL-2, immunosuppressant Transplantation Respiratory infection Synciitial Virus Arthritis HER-2/neu (EGF2) breast cancer CD33 leukemia (AML) Leukemia Lymphoma Arthritis IgE asthma Lymphoma Psoriasis EGF-R colon cancer VEGF colon cancer

8. Monoclonal antibody generation • - Cells needed myeloma cells, mouse spleen cells • - antigen administration Kohler and Milstein • - hybridoma formation via cell fusion • selection mutants required (hprt- usually) • - antibody generation cDNA cloning • - engineered MAbs expression vectors • - refinement chimeric, humanized, human

9. 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

10. Reduced myeloma hybrid Cesar Milstein Georges Kohler Unreduced myeloma hybrid Kohler, G., and C. Milstein (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495-497. 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. Isoelectric focusing immunoglobulins made in hybridoma cells

11. 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 Soluble CD4 (HIV receptor) fused to IgG2. Tetrameric (4 V-regions replaced) Reduced Fc function (since IgG2 < IgG1), Better half-life Progenics

12. Phage display selection of scFv Source of sequence: PCR from genome or mRNA, add randomization (doped synthesis) Single chain antibodies (scFv) Ag binding site 15 AA linker

13. Protein Glycosylation Stanley, P. 1989. Chinese hamster ovary cell mutants with multiple glycosylation defects for production of glycoproteins with minimal carbohydrate heterogeneity. Mol Cell Biol9: 377-383. 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 Biotechnol17: 176-180. Review: Grabenhorst, E., Schlenke, P., Pohl, S., Nimtz, M., and Conradt, H.S. 1999. Genetic engineering of recombinant glycoproteins and the glycosylation pathway in mammalian host cells. Glycoconj J16: 81-97. 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

15. = Penta- saccharide common core = Diantennary With bisecting GlcNAc With fucosylated core Triantennary (also tetra-antennary) All shown here, N-linked (to amide N of Asn in N-X-S or N-X-T) Substantial in size Carbohydrates attached to loops or near termini Also O-linked, to ser or thr (hydroxyl on side chain)

16. 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.

17. 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) • afucosylated

18. neuraminic acid – one of the sialic acids = : both terms are used, confusedly NAcNeu: Carboxyl (acid) Glycerol moiety mannose Acetylated amino group deoxy

19. Glycosylation pattern affects signaling, for: Delivery to the right cell receptor for activity Clearance rate Microheterogeneity: Lots of isoforms, naturally No apparent 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

20. 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)

21. Modifying glycosylation • Add or subtract sites to your favorite protein (cis) • Change the general glycosylation phenotype of the host cell (trans) 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

22. Modifying glycosylation • Add or subtract sites to your favorite protein (cis) • Change the general glycosylation phenotype of the host cell (trans) 2. Clone enzyme genes:Glycosyl transferases, mostlyAlso 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).

23. Isolate mutant mammalian cell lines deficient in specific glycosylation enzymes Stanley: Isolation of multiply mutated glycosylation mutants by selecting for lectin resistancei Lectins = carbohydrate-binding proteins Plant lectins used mostly here (but occur widely) 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 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 for 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 Pam Stanley

24. Stanley, P. 1989. Chinese hamster ovary cell mutants with multiple glycosylation defects for production of glycoproteins with minimal carbohydrate heterogeneity. Mol Cell Biol9: 377-383. transport to Golgi 5 Golgi glucose Exploits hypersenstivity to select against certain phenotypes.

25. mutants

26. Sequential mutagenesis and selections to isolate mutliply-mutated glycosylation mutants

27. Predicted alteredglycosylation statesin various mutants

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 Biotechnol17: 176-180. Target here (bisecting NAcG) Presence of the bisecting NaG 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.

29. TARGET CELL (Killer T-cell) Genentech Commercial MAb injected as a therapeutic T-cell surface receptor binds Fc region of antibody molecule(Fc gammaR)

30. Getting CHO cells to make more bisected oligosaccharide in the Fc region of MAbs to better activate antibody-dependent cellular cytotoxicity (ADCC) GnT III = glycosyltransferase in question Methods: Vectors (8) : tTa neoR rat GnTIII cDNA+myc+histag no introns that into tet promoter vector Pur H-chain cDNAs (CMV + bGH pA + SV40neo) synthetic leader L-chain cDNAs (CMV + bGH pA + SV40neo) synthetic leader zeoR Tet-driven beta-galactosidsae Transfections (4): tTA + neo, transient tet-beta-gal, GnTIII+pur, H+L+zeo Westerns Mass Spec, incl. enzyme digestions: sialidase; peptide N-glycosidase F (4 vs. 5 hexoses???) ADCC (dye retention/release, neuroblastoma cells)

31. Target here (bisecting NAcG) Variably present Tet-off system Transient transfection of GnTIII into tTA-bearing CHO cells (western blot) Permanent transfectant for tTA and GnTIII tTA = Tetracycline responsive TransActivator protein

32. Tet= 2000 Tet= 60 Tet= 30 Tet= 15

33. Tet= 2000 Mass spec products indicating the presence of the bisecting NAcG (in dashed boxes) Tet= 60 Tet= 30 Tet= 15

34. ADCC assay ADCC correlates with bisected complex content Tet induction of GnTIII No induction of GnTIII

35. Result: ADCC efficiency followed proportion of oligosaccharide with bisected sugar Bisecting sugar:15%  45% ADCC: 25  50% Missing: Zero bisection control CHO cells are supposed to LACK GnTIII and Westerns show ~0 rat GnTIII at 2000 ug/ml tetracycline Yet backgrounds of bisecting sugar are high. OK for ADCC, but Mass Spec data ……. Extensions? Try untransfected CHO? Westerns lying? ( <30 ug.ml tet  death …too much enzyme?) Good example of enzyme engineering. Can still be optimized. Use a constitutive promoter, try different version to find the best using ADCC as the assay Check dependence on Ig production level.

36.  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

37. DT= diphtheria toxin gene, Kills if intergated via non-homologous recombination Lox sites Double knock-out strategy for FUT8 an alpha-1,6,fucosyl transferase Isolate CHO cDNA using mouse sequence data fro primers Use CHO cDNA to isolate CHO genomic fragments from a commercial lambda library K.O. exon 1 translation start Homology regions For hemizygote: 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  10 double KO homozygotes. Remove drug resis. genes by Transient trasnfection with Cre recombinase Note: 10’s of thousands of PCRs performed to screen for homologous recomb., using 96-well plates

38. Double knockout evidence Orginal KO’d genes have a 1.5 kb insertion (Southern blot) mRNA has 200 nt deletion (RT-PCR

39. Use of a fluoresceinated 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 +/- FUT8 +-/-

40. Rituxan (anti-CD20) produced in FUT -/- cells does not contain fucose(HPLC analysis) Digestion all the way to monosaccharides

41. In ADCC, FUT8-/- anti-CD20 >> Rituxan Binding to CD20 membranes FUT8-/- anti CD20 = Ritxuan Anti-CD20 from a partially FUT-deficient rat cell line Rat line FUT-/-’s Complement-mediated cell toxicity is the same for FUT8-/- and Rituxan Rituxan = commercial product, 98% fucosylated

42. 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)?

44. Quality-Control System for Protein Folding in the ER. Folded protein cannot get re-glycosylated. Only deglycosylated proteins move from ER to Golgi. Biochemistry. 5th edition Berg, Jeremy M.; Tymoczko, John L.; and Stryer, Lubert.

45. LexGene Trap Zambrowicz BP, et al. Disruption and sequence identification of 2,000 genes in mouse embryonic stem cells. Nature. 1998;392(6676):608-11.