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Monoclonal Antibodies MAbs

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Title: Monoclonal Antibodies MAbs


1
  • Monoclonal Antibodies (MAbs)
  • Antibodies (Abs). Also known as immunoglobulins
    (Ig).
  • Comprised of 2 heavy chains and 2 light chains
  • Monoclonal Abs bind specifically to a single
    site (epitope) on a particular antigen
  • - Abs are produced by B lymphocytes
  • Because of their specificity and ease of
    generation, they are extensively used as
    therapeutics (passive immunotherapy) and as
    diagnostic and research tools
  • -They can be generated in large (unlimited)
    amounts in culture

2
Antibodies are made by B-cells
Red blood cells (RBCs)
B cells develop in the bone marrow ?
hematopoietic stem cells and
lymphoid stem cells
? T-cells and B-cells progenitor pro-B
cell (B220) precursor pre-B cells
heavy-chain rearranged immature B cell
IgM light-chain rearranged mature B cell IgM
IgD an antigen encountered in spleen or lymph
nodes then goes to peripheral circulation Termin
ally differentiated cell plasma cell,
periphery, Ig secretor (IgG, IgM, some
others) Each immunocyte (and its offspring)
synthesize only a single type of Ig, and use
only one of the two alleles available (allelic
exclusion)
Immunocytes at different stages or of different
types are often characterized by specific cell
surface proteins, often acting as antigens
See Strachan and Read, pp. 307-311
3
Domain structure of an immunoglobulin molecule
disulfide bonds
4
Heavy chain blue Light chain pink
5
Laboratory fragmentation of antibodies
6
Ig molecule showing polarity, disulfides,
carbohydrate
7
Fc functions
Opsonization Complement activation Antibody-depe
ndent cell-mediated cytotoxicity
(ADCC) Transcytosis
8
Soluble, circulating, most abundant
Most membrane-boundHigh avidity via multivalency
Secreted in extracellular fluids (mucus, tears,
saliva)
Targeted to parasites, allergic reaction
IgA dimer
?
9
Multigene organization of Ig genes light
chains V, J (variable) and C (constant) heavy
chain V, D, J, (variable) C (constant) Mechanis
m of Ab gene rearrangement Recombination signal
sequences (RSS)flank V, D, J gene
segments V-RSS------RSS-D-RSS---------RSS-J
10
IgGkappa gene rearrangement
SOMATIC HYPERMUATION
LIGHT CHAIN
(J)
(J)
SPLICING
(J)
(D,J)
SPLICING
(D,J)
SOMATIC HYPERMUATION
HEAVY CHAIN
11
Choice of Constant region exons (class switching)
takes place via DNA recombination (below) and
alternative splicing of pre-mRNA
Immunobiology, Charles Janeway, Paul Travers,
Mark Walport, Mark Shlomchik
12
Different constant regions can be chosen via
alternative pre-mRNA splicing
Immunobiology, Charles Janeway, Paul Travers,
Mark Walport, Mark Shlomchik
13
Alternative splicing within a group of Constant
region exons yields two forms of IgM
Developmental Biology, Eighth Edition, Scott F.
Gilbert
14
Different classes of Igs have different properties
15
Fc functions
Opsonization Direct uptake of bacteria coated
with antibody molecules Complement activation
Activated complement proteins lyse cells by
making holes in their mebranes(e.g.
bacteria) Antibody-dependent cell-mediated
cytotoxicity (ADCC) Killer T-cells use
antibodies on their surface to target cells with
an antigen and kill them. Transcytosis Antibody-
antigen complexes are taken up (endocytosed) on
one side of an epithelial cell and directed to
the other side, where they are exocytosed
Fc
16
Antibodies can participate in host defense in
three main ways
17
ADCC antibody-dependent cell-mediated
cytotoxicity
Many anti-cancer MAb therapies are based on ADCC
18
NK cell
Genentech
ADCC antibody-dependent cell-mediated
cytotoxicity
Fc region
19
MAb therapy targets Inflammation Autoimmune
disease Graft rejection Cancer Viral infection
20
Therapeutic strategies MAbs straight MAbs fused
to other protein binders (e.g., soluble
receptors) to increase avidity and/or effect
ADCC MAbs fused to cytotoxic agents (toxins,
radionuclides) Toxins ricin (stops protein
synthesis) calicheamicin (DNA breaks) Radionucl
ides 90Y yttrium 111I indium
21
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
22
  • 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

23
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
24
Reduced S-S bonds myeloma hybrid
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 of immunoglobulins made
in hybridoma cells
25
  • 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 3559205
  • Solutions via recombinant DNA genetic engineering
  • Chimeric mouse-human antibodies mouse V regions
    fused to Hu C-region
  • 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 complementarity-determining region
26
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 molecules to which HIV
attaches on T-cells Soluble CD4 (HIV receptor)
fused to IgG2. Tetrameric (all 4 V-regions
replaced) Reduced Fc function (since IgG2 lt
IgG1), Better half-life (Recently replaced by a
MAb (PRO 140) targeting the CCR5 cell surface
protein, required for viral entry) Progenics
TNF tumor necrosis factor)
27
Single chain antibodies (scFv)
28
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 A
physical link a nucleic acid sequence (here, DNA)
to the phenotype (e.g., binding to another
proteina) of a protein coded by that nucleic acid
29
Phage display selection of scFvs (single-chain
F-variable regions) Source of sequence PCR from
genome or mRNA, add randomization (doped
synthesis)
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 587(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
594(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, S.,
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.
31
Two types of protein glycosylation
32

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
Fucose
Also O-linked, to ser or thr (hydroxyl on side
chain)
33
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.
34
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 fucosyla
ted bisecting GlcNAc Blood (made in
liver) diantennary NAcNeu (sialated sialic
acid) afucosylated
Sialic acid structure see next graphic
35
neuraminic acid one of the sialic acids
both terms are used, confusedly
NAcNeu
Carboxyl (acid)
Glycerol moiety
mannose
Acetylated amino group
deoxy
36
Glycosylation pattern affects signaling,
for Delivery to the right cell receptor for
activity 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
37
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)
38
  • Modifying glycosylation
  • 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
    proteins structure)
  • Works
  • Change the general glycosylation phenotype of the
    host cell (trans)
  • E.g., Pam Stanley lectin-resistant mutants

39
  • Modifying glycosylation
  • Add or subtract sites to your favorite protein
    (cis)
  • Change the general glycosylation phenotype of the
    host cell (trans)

2. Clone enzyme genesGlycosyl transferases,
mostlyAlso some synthetases (e.g., NAcNeu) Can
be complexe.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) ExperimentOver-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).
40
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) 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
41
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.
42
TARGET CELL
(Killer T-cell)
Genentech
Commercial MAb injected as a therapeutic
T-cell surface receptor binds Fc region of
antibody molecule(Fc gammaR)
43
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
44
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
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
transfection with Cre recombinase
Note 10s of thousands of PCRs performed to
screen for homologous recomb., using 96-well
plates
45
Double knockout evidence
After Cre treatment
Orginal KOd genes have a 1.5 kb
insertion (Southern blot)
mRNA has 200 nt deletion (RT-PCR
46
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 /-
FUT8 -/-
47
Rituxan (anti-CD20) produced in FUT -/- cells
does not contain fucose(HPLC analysis)
Digestion all the way to monosaccharides
Missing d - g
48
In ADCC, FUT8-/- anti-CD20 gtgt Rituxan
Binding to CD20 membranes 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
49
Very laborious, but apparently a big
payoff. Better selection? Why not use the
fluorescent LCA to select for the FUT8 KOs along
with G418 resistance(double sequential
selection)?
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