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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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slide1

MAbs continued

Antibodies can participate in host defense in three main ways

slide2

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.

slide3

TARGET CELL

(Killer T-cell)

Genentech

Commercial MAb injected as a therapeutic

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

slide4

MAb therapy targets

Inflammation

Autoimmune disease

Graft rejection

Heart disease (thrombosis)

Cancer

Viral infection

slide5

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

slide6

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

slide7

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

slide8

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
slide9

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

slide10

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

slide11

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

slide12

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

slide13

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

slide15

=

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)

slide16

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.

slide17

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
slide18

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

NAcNeu:

Carboxyl (acid)

Glycerol moiety

mannose

Acetylated amino group

deoxy

slide19

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

slide20

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)

slide21

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

slide22

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).

slide23

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

slide24

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.

slide26

Sequential mutagenesis and selections to isolate mutliply-mutated glycosylation mutants

slide28

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.

slide29

TARGET CELL

(Killer T-cell)

Genentech

Commercial MAb injected as a therapeutic

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

slide30

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)

slide31

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

slide32

Tet= 2000

Tet= 60

Tet= 30

Tet= 15

slide33

Tet= 2000

Mass spec products

indicating the presence

of the bisecting NAcG

(in dashed boxes)

Tet= 60

Tet= 30

Tet= 15

slide34

ADCC assay

ADCC

correlates

with bisected

complex content

Tet induction of GnTIII

No induction of GnTIII

slide35

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.

slide36

 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

slide37

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

slide38

Double knockout evidence

Orginal KO’d genes have a 1.5 kb insertion

(Southern blot)

mRNA has 200 nt deletion

(RT-PCR

slide39

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 +-/-

slide40

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

Digestion all the way to monosaccharides

slide41

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

slide42

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)?

slide44

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.

lexgene trap
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.