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
B cells develop in the bone marrow?hematopoietic
stem cells and lymphoid stem cells 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 matured B
cell IgM IgD an antigen encountered in
spleen or lymph nodes then goes to peripheral
circulation Terminally differentiated cell
plasma cell, periphery, Ig secretor (IgG, IgM,
some others)
Immunocytes at different stages or of different
types are often characterized by characteristic
specific cell surface proteins, often acting as
antigens
Each immunocyte (and its offspring) synthesize
only a single type of Ig, and use only one of
the two alleles available (allelic exclusion)
For a summary of the immune system see Strachan
and Read, pp. 119-131
3
Domain structure of an immunoglobulin molecule
Heavy chain
Light chain
disulfide bonds
C constant regions V variable regins (antigen
binding) H heavy chain L light chain
4
Heavy chain blue Light chain pink
5
Laboratory fragmentation of antibodies
6
A schematic view of an Ig molecule showing
polarity, disulfides, carbohydrate
CHO carbohydrate
7
Fc functions
Fc functions
Opsonization Complement activation Antibody-depe
ndent cell-mediated cytotoxicity
(ADCC) Transcytosis
Fc
8
Disulfide bond
IgM pentamer
J-chain
Secretory IgA dimer
9
Multipart organization of Ig genes light
chains V, J (variable) and C (constant) heavy
chain V, D, J, (variable) C (constant) Mechanis
m of Ig gene rearrangement Recombination signal
sequences (RSS)flank V, D, J gene
segments V-RSS------RSS-D-RSS---------RSS-J
10
IgGkappa gene rearrangement
Light chain genesis
DNA
SOMATIC HYPERMUTATION
(J)
(J)
SPLICING
(J)
(D,J)
SPLICING
(D,J)
SOMATIC HYPERMUTATION
Heavy chain genesis
DNA
95Vh, 30Dh,5Jh, 11Ch over 1.4 Mb
L leader sequence, signal for secretion
11
Alt. splicing
IgD
Adapted from Janet Stavnezer http//www.umassmed.
edu/faculty/show.cfm?start0faculty300
12
Also Alternative splicing within a group of
Constant region exons yields two forms of IgM
pA
pA
Developmental Biology, Eighth Edition, Scott F.
Gilbert
13
Different classes of Igs have different properties
14
Fc functions
Opsonization Direct uptake into macrophages of
bacteria coated with antibody molecules Complemen
t activation Activated complement proteins
lyse cells by making holes in their mebranes
(e.g. bacteria) 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
Antibody-dependent cell-mediated cytotoxicity
(ADCC) Cells with a surface antigen are coated
with antibodies that bind via their Fab region.
Then killer T-cells use Fc receptors on their
surface to recognize these cells and kill them.
Fc
15
Antibodies can participate in host defense in
several ways
Also ADCC
16
ADCC antibody-dependent cell-mediated
cytotoxicity
NK cell
Fc receptor
Fc
NK cells natural killer T-cells
17
NK cell
Genentech
Fc region
ADCC antibody-dependent cell-mediated
cytotoxicity
18
Antibody generation
T-cells cell mediated immune reactions and B-cell
s ? secreted antibodies
2002 Molecular Biology of the Cell by Alberts,
Johnson, Lewis, Raff, Roberts, and Walter.
19
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 stimulated. 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.
20
The antibody secreting effector cells terminally
differentiate (die) but their sister memory cells
live on to generated an amplified response upon a
second exposure to antigen
1st exposure
Effector cells
Memory cells
2nd exposure
2002 Molecular Biology of the Cell by Bruce
Alberts, Alexander Johnson, Julian Lewis, Martin
Raff, Keith Roberts, and Peter Walter.
21
2002 Molecular Biology of the Cell by Bruce
Alberts, Alexander Johnson, Julian Lewis, Martin
Raff, Keith Roberts, and Peter Walter.
22
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)
TG 6-thioguanine Hat hypoxanthine,
amethopterin, and thymidine
23
Immunize with antigen X
Monoclonal antibody generation
Hprt- myeloma cells 6-TG-resisatnt HAT-
(HAT)
Cell fusion
Plate among many wells for supernantant testing
Selection in HAT
Screen for secreted anti-X antibody
Plate positives at low density (1/well) for
cloning
Positive clones provide a continuing source of
anti-X antibody
24
MAb therapy targets Inflammation Autoimmune
disease Graft rejection Cancer Viral infection
25
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) Radionucl
ides 90Y yttrium 111I indium
26

• 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)
• Further development
• - antibody generation cDNA cloning from hybridoma
• - engineered MAbs expression vectors
• refinement 2nd generation antibodies, in vitro
• Solve problems of using mouse
  antibodies in humans

27

• 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 as source of
  spleen cells (Medarex, Abgenix, Kirin)

CDR complementarity-determining region
28
MAb Fusion Proteins With 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
Example, still experimental Anti-HIV drug PRO
542 Uses soluble form of the CD4, the molecule
to which HIV attaches on T-cells Aim block the
viral surface protein that binds CD4 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
29
Single chain antibodies (scFv)
30
Phage display to isolate functional V-regions
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 of a similar kindA 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
Commonly used phage m13, filamentous, infects
E. coli
Phage coat protein
the protein
the DNA(inside)
Panning
E.g., for a SC Ab, coat the dish with Ag
Protein displayed in the phage coatCan screen
108 phage
31
Phage display selection of scFvs (single-chain
variable regions) Source of sequence PCR from
genome or RT-PCR from mRNA, add randomization
(doped synthesis).
Repeat, to reduce background
Wash
or
Elute, re-infect
PCR rescue scFv DNA
Clone (plaque)