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MAbs continued. Antibodies can participate in host defense in three main ways . ADCC = antibody-dependent cell-mediated cytotoxicity. NK = Natural killer cells (T-cell class) FcgammaRIII = Fc receptor on NK cell surface.

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MAbs continued

Antibodies can participate in host defense in three main ways


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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.


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TARGET CELL

(Killer T-cell)

Genentech

Commercial MAb injected as a therapeutic

T-cell surface receptor binds Fc region of antibody molecule(Fc gammaR)


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MAb therapy targets

Inflammation

Autoimmune disease

Graft rejection

Heart disease (thrombosis)

Cancer

Viral infection


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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


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  • 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


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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


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  • 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


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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


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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


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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


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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


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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


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=

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)


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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.


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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


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    neuraminic acid – one of the sialic acids = : both terms are used, confusedly

    NAcNeu:

    Carboxyl (acid)

    Glycerol moiety

    mannose

    Acetylated amino group

    deoxy


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    Glycosylation pattern affects signaling, for: are used, confusedly

    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


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    Genetic engineering of glycosylation to: are used, confusedly

    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)


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    • Modifying glycosylation are used, confusedly

    • 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


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    • Modifying glycosylation are used, confusedly

    • 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).


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    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


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    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.


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    mutants multiple glycosylation


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    Sequential mutagenesis and selections to isolate mutliply-mutated glycosylation mutants


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    Predicted altered mutliply-mutated glycosylation mutants glycosylation statesin various mutants


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    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.


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    TARGET CELL Bailey, J.E. 1999.

    (Killer T-cell)

    Genentech

    Commercial MAb injected as a therapeutic

    T-cell surface receptor binds Fc region of antibody molecule(Fc gammaR)


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    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)


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    Target here the Fc region of MAbs to better activate antibody-dependent cellular cytotoxicity (ADCC)(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


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    Tet= 2000 the Fc region of MAbs to better activate antibody-dependent cellular cytotoxicity (ADCC)

    Tet= 60

    Tet= 30

    Tet= 15


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    Tet= 2000 the Fc region of MAbs to better activate antibody-dependent cellular cytotoxicity (ADCC)

    Mass spec products

    indicating the presence

    of the bisecting NAcG

    (in dashed boxes)

    Tet= 60

    Tet= 30

    Tet= 15


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    ADCC assay the Fc region of MAbs to better activate antibody-dependent cellular cytotoxicity (ADCC)

    ADCC

    correlates

    with bisected

    complex content

    Tet induction of GnTIII

    No induction of GnTIII


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    Result: the Fc region of MAbs to better activate antibody-dependent cellular cytotoxicity (ADCC)

    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.


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     Biotechnol Bioeng. 2004 Sep 5;87(5):614-22 the Fc region of MAbs to better activate antibody-dependent cellular cytotoxicity (ADCC)

    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


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    DT= diphtheria toxin gene, the Fc region of MAbs to better activate antibody-dependent cellular cytotoxicity (ADCC)

    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


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    Double knockout evidence the Fc region of MAbs to better activate antibody-dependent cellular cytotoxicity (ADCC)

    Orginal KO’d genes have a 1.5 kb insertion

    (Southern blot)

    mRNA has 200 nt deletion

    (RT-PCR


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    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 +-/-


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    Rituxan (anti-CD20) produced in FUT -/- cells does not contain fucose(HPLC analysis)

    Digestion all the way to monosaccharides


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    In ADCC, FUT8-/- anti-CD20 >> Rituxan contain fucose

    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


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    Very laborious, but apparently a big payoff. contain fucose

    Better selection?

    Why not use the fluorescent LCA to select for the FUT8 KO’s along with G418 resistance(double sequential selection)?


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    Quality-Control System for Protein Folding in the ER. contain fucose

    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.


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    LexGene Trap contain fucose

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


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