Transcript Monoclonal Antibodies

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
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+)
Immunocytes at different stages or of
different types are often characterized by
characteristic specific cell surface
proteins, often acting as antigens
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)
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
Domain structure of an immunoglobulin molecule
Heavy chain
Light chain
V-region
}
C = constant regions
V = variable regins (antigen binding)
H = heavy chain
L = light chain
}
Fab
Fragment ,
antigen binding
disulfide
bonds
Fc
Fragment , crystallizable
Heavy chain = blue
Light chain = pink
Laboratory fragmentation of antibodies
A schematic view of an Ig molecule showing polarity, disulfides, carbohydrate
Greek letters refer to
different combination
of constant regions
Complementarity
determining
Region = “CDR”
Hypervariable
region
CHO = carbohydrate
Fc functions
Fc functions
Opsonization
Complement activation
Antibody-dependent
cell-mediated
cytotoxicity (ADCC)
Transcytosis
Fc
Disulfide bond
IgM
pentamer
J-chain
Secretory IgA dimer
Multipart organization of Ig genes
–light chains: V, J (variable) and C (constant)
–heavy chain: V, D, J, (variable) C (constant)
Mechanism of Ig gene rearrangement
Recombination signal sequences (RSS)–flank V, D, J gene segments
–V-RSS------RSS-D-RSS---------RSS-J
IgGkappa gene rearrangement
76 Vk, 5 Jk, 1 Ck over 2 Mb
Light
chain
genesis
DNA
+ SOMATIC
HYPERMUTATION
(J)
(J)
SPLICING
(J)
(D,J)
SPLICING
(D,J)
Heavy
chain
genesis
+ SOMATIC
HYPERMUTATION
L = leader sequence, signal for secretion
DNA
95Vh, 30Dh,5Jh, 11Ch over 1.4 Mb
Alt.
splicing
IgD
Adapted from Janet Stavnezer http://www.umassmed.edu/faculty/show.cfm?start=0&faculty=300
Also:
Alternative splicing within a group of Constant region exons yields two forms of IgM
pA
Developmental Biology, Eighth Edition, Scott F. Gilbert
pA
Different classes of Igs have different properties
Fc functions
Opsonization:
Direct uptake into macrophages of bacteria coated with antibody molecules
Complement 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
Antibodies can participate in host
defense in several ways
Also ADCC
ADCC = antibody-dependent cell-mediated cytotoxicity
NK
cell
Fc
NK cells = natural killer T-cells
Fc receptor
NK cell
Genentech
Fc region
ADCC = antibody-dependent cell-mediated cytotoxicity
Antibody generation
2002 Molecular Biology of the Cell
by Alberts, Johnson, Lewis, Raff,
Roberts, and Walter.
T-cells: cell mediated immune reactions
and
B-cells:  secreted antibodies
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.
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
Memory cells
2nd exposure
2002 Molecular Biology of the Cell by Bruce Alberts,
Alexander Johnson, Julian Lewis, Martin Raff, Keith
Roberts, and Peter Walter.
Effector cells
2002 Molecular Biology of the Cell by
Bruce Alberts, Alexander Johnson, Julian
Lewis, Martin Raff, Keith Roberts, and
Peter Walter.
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
Immunize with antigen X
Monoclonal antibody generation
Hprt- myeloma cells
6-TG-resisatnt
HAT-
(HAT)
Cell fusion
Selection in HAT
Plate among many wells for
supernantant testing
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
MAb therapy targets
Inflammation
Autoimmune disease
Graft rejection
Cancer
Viral infection
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)
Radionuclides:
90Y = yttrium
111I = indium
Monoclonal antibody generation
- Cells needed
- antigen administration
- hybridoma formation
- selection
myeloma cells and mouse spleen cells
Kohler and Milstein
via cell fusion
mutants required (myeloma hprt- usually)
Further development:
- antibody generation
- engineered MAbs
- refinement
cDNA cloning from hybridoma
expression vectors
2nd generation antibodies, in vitro
Solve problems of using mouse antibodies in humans
Problems of mouse MAbs
1) Fc portion limited in its ability to interact with Fc receptors of
human cells.
2) Lower serum half-life
3) Development of human anti-mouse antibodies (HAMA)
A) Retreatment results in allergy or anaphylactic shock
B) Retreatment is less effective
Breedveld, Lancet 2000
355:9205
Solutions via recombinant DNA genetic engineering :
1) Chimeric mouse-human antibodies: mouse V regions fused to Hu C-region
2) Humanized mouse antibodies, Parts of V-region from human interspersed with
mouse CDR V-regions
3) Human antibodies (fully), via transgenic mice carrying human immunoglobulin
genes as source of spleen cells
(Medarex, Abgenix, Kirin)
CDR = complementarity-determining region
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
Single chain antibodies (scFv)
Ag
binding
site
15 AA
linker
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 kind:
A 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 coat
Can screen 108 phage
Phage display selection of scFv’s (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)