Antibodies Flashcards
Alain Beck et al (2010) Strategies and challenges for the next generation of therapeutic antibodies. Nature Reviews Immunology, 10, 345-352 Ahmad P et al (2012) Role of transgenic plants in agriculture and biopharming., Biotechnol Adv., 30, 524-40 Teillaud Jean-Luc (2012). From whole monoclonal antibodies to single domain antibodies: Think small. Methods in Molecular Biology, 911, 3-13 Cioffi et al., (2012) EpCAM/CD3 Bispecific T cell Engaging Antibody MT110 Eliminates Primary Human Pan
Ab Functions
Bind specifically to molecules on pathogen/antigen that created immune response
recruit cells and molecules to destroy pathogen/antigen once bound
Structure
each chain series of similar sequences, each repeat corresponds to domain
CH2 domains not interact due to
carbohydrate side chains
Domain structure
two anti-parallel arrays of Beta strands forming two beta pleated sheets
VH +VL, CH + CL held together by hydrogen bonds and non-hyrdophobic interactions
The most variable regions and structure
Highly variable regions 1,2 and 3, 3 being the most variable
form 3 loops and antigen binding site
3 loops are complimentary determining regions CDR1,2+3
Regions between HV regions -
framework regions FR1,2,3+4
form Beta sheets
Polyclonal antibody
different antibodies even against same antigen
from immunised animal - immunoglobulin isolated
monoclonal antibody
uniform population
same binding specificity, affinity and biological activity, produced by identical hybridoma cells
Hybridoma cells
fusion of immune lymphocytes with myeloma cells - antibody secreting and long term survival potential
Monoclonal antibodies - Immunisation protocol
Most commonly produced monoclonal antibodies are in mouse
Purity of immunogen not important as selection of specific antibody secreting cell lines anmals must be boosted 4 days before fusion - lymphocytes at peak proliferation
source of lymphocytes - spleen or lymph nodes
Myeloma cell properties required
enzyme deficiency (e.g. HGPRT)/unique chemical sensitivity to enable selection of fused from unfused
not secrete immunoglobulin
(usually easy to grow in tissue culture)
Fusion protocol
single cell suspension immune splenocytes and myeloma cells mixed and pelleted, PEG is added to the cells slowly - promotes cell fusion
PEG diluted in medium, cells centrifuged and resuspended in medium
Nucleic acid synthesis routes
de novo, if not then salvage
purine synthesis- uses hypoxanthine and guanine by HGPRT (hypoxanthine guanine phosphribosyl transferase) forming guanine acid ribose phosphate and ionsinic-ribose phosphate
pyrimidine synthesis - uses thymidine by thymidine kinase to form thymidine monophosphate
HAT medium contains
aminopterin - antibiotic blocks de novo
hypoxanthine and thymidine - for salvage
enzymes not present in myleoma cells
Only hybrid survive
B cells have finite lifespan
myeloma cells have no HGPRT
Screening Test
ELISA - as similar to final assay
Cloning protocol
Limited Dilution
supplemented with feeder cells (peritonal macrophages, splenocytes) provide growth factors
supernatant tested for antibodies
Crypreservation
store in liquid nitrogen
Polyclonal antibody qualities (6)
cross react limited supply choice of species precipitate best with pure antigen easy to screen
Monoclonal antibody qualities (6)
specific unlimited limited species (rat, mouse) not precipitate not need purified antigen need good screening
HAMA
Human anti mouse antibody
mouse antibodies only 60-70% similar
Possibly prevent by
replacing non-specific part with human part
Human antigen binding fragments created from
combinational libraries as bacteria express Fab and Fvs
Ab ideal for recombination as
each domain is coded for by different exon - chimeric
Detect Ig genes with
Southern Blotting
Chimeric antibodies exhibit
reduced immunogenecity
Infliximab/Remicade used to treat
Inflammatory diseases - crohns and rheumatoid arthritis
Grafting CDR regions from specific rodent monoclonals
transfer of specific binding and reduced antigenicity
Framework structure of variable region
can help determine shape of CDR loops
designed by crystallography
Omalizumab (Xolair) structure and use
humanised, used for severe allergic asthma
Humanised Antibodies development
Combine monoclonal murine CDR with human framework
Superhumanised antibodies
incorporate human regions into CDR
Fv fragment structure and function
bind antigen, heterodimer of VH and VL
unstable as non-covalently linked chains dissociate easily
Single chain fv fragment (scfv)
high affinity for antigen and expressed in many hosts
scFv linker
domains joined by hydrophilic, flexible peptide - improves expression and folding efficiency
15AA
monomeric scFv
compexed into dimers (diabodies) etc.
same specificity - bivalent
different - bispecific - novel functions
Bispecific antibodies construction
chemical recombination
cell fusion
genetic manipulation
Chemical recombination
Fc removed by pepsin digestion
Interchain disulphide removed by reducing agent - 2 mercaptoethanol
fragments purified by chromatography
Chemical recombination - rejoining
random - oxidative reformation interchain disulphide of hinge
specific by thioester bonding with specific linker
BiTE
Bispecific T cell engaging antibody
one binds to target epitiope on target cell and other binds effector T cell
BiTE blocking
block receptors on cell surface
EGFR and insulin-like growth receptor - inhibit tumour growth
Trispecific better… (2)
tighter binding
enhanced effector function
Bispecfic future tech (5)
DART Modular Ab technology Bicycle Tech Dual targeting domains Chemical generation
Single domain antigen binding fragments (dAbs) VhS
good affinity
but sticky and difficult to produce in soluble form
Heavy chain antibodies (hcAb) IgG
no light chain
sharks, camelidae
hcAb structure
95kDa
single heavy chain variable domain VHH/VNAR
followed immediately by hinge region then CH2/CH3?5xCHNAR
hcAb affinity and application
similar to Fab and scFv
easier to produce soluble
hcAb application (6)
cancer imaging and immunotherapy cytokine neutralisation competitive enzyme inhibition block virus secretion trypanosome drug delivery malaria diagnosis
Camelisation
VL side replaced with VHH-like residues
increase solubility and non-specific binding
Plant antibodies
Transgenic plants
Ab against dental caries, RA, cholera etc.
CaroRX
sIgA - dental caries
oral antiseptic then antibody
Intrabody
Intracellular antibody
expressed within designated cell compartment
Intrabody function
interruption/modification of target protein functions
scFv, single domain intrabodies intradiabodies (bispecific)
Intrabody Use (5)
sequestration protein from compartment mediate enzyme function (block/change conformation) disruption transduction induction apoptosis ubiquitin-proteosome degradation
Intrabody construction
clone V region into vector (retro/adeno)
Intrabody treatement (3)
Huntingtons
Cancer - alter neoplastic properties
Interruption HIV lifecycle
Phage Display
Foreign gene sequence spliced into gene sequence of coat protein, AA sequences fused - hybrid protein
hybrid coat protein incorporated into phage particles and displayed on the outer surface
Phage Display requirements
clone gene into filamentous bacteriophage vector with ssDNA and can infect male E.coli (pili)
Filamentous bacteriophage attachment
N-terminal domain of pIII to tip of the pilus
Phage panning
antibody gene fused wit cpIII gene, it is displayed on surface and is selected when it binds to its antigen where it can be eluted
Uses of phage produced Antibodies
ELISA
Soluble antibody fragments
Ribosome Display
cell free, in vitro selection of proteins and peptides from large libraries
coupling protein to its mRNA by forming stable protein-ribosome-mRNA (PRM) complex
enable formation and amplification
Ribosome stalling (2)
- Add antibiotics rifampicin/chloramphenicol (prokaryotes) or cyclohexamide (eukaryotes ) to halt translation
- Deletion of stop codon normally recognised by release factors that trigger removal peptide form mRNA
Prokaryotic Ribosome Display - PRM complexes - method
- polysome complex, where complexes were captured with immobilised antibody
- EDTA released bound mRNA, then —> cDNA
Eukaryotic Ribosome Display technique
ARM - antibody-ribosome-mRNA complex been developed
captured on antigen coated magnetic beads
mRNA not needed to be dissociated
Ribosome display efficient at selecting
high affinity scFv
Genetically Engineered Mice produce human Ab
Denosumab anti-RANKL antibody - treat osteoporosis, bone metastases and induced bone loss
TNFalpha in RA
formation osteoclasts - bone breakdown
proliferation synoviocytes - joint inflam
expression collagenase by chondrocytes - joint/cartilage destroy
anti-TNFalpha effect
reduce proinflam cytokines - IL-1,6,8 and GM-CSF
Chimeric anti-TNFalpha
infliximab/remicade ) with methotrxate
IL-1r blockade
Anakinra (soluble IL-1Ra)
TNFR-Fc fusion protein
Etanercept
SE RA Antibodies
Tuberculosis
QUALY
Quality-adjusted life year
value for money
no. years of life added - 1.0 = fully healthy year
Herceptin target
HER-2 - expressed highly in cancer
Herceptin action
prevents transmission of growth signal to nucleus and stimulated ADCC
Rituximab target
targets CD20 in Non-Hodgkins Lymphoma
on cancerous B cells
Rituximab action
stimulates Antibody mediated cellular cytotoxicity
and complement mediated cytotoxicity
Gemtuzimab-ozogamacin target
CD33 in acute myeloid leukaemia on leukaemic cells
Gemtuzimab-ozogamacin action
bind CD33 and internalised ozogamacin released
into lysosomes of cells
binds DNA and strand breaks and apoptosis
Ibritumomab-tiuxetetan target (IgG1 90Y conjugate)
bind CD20 on non-hodgkins lymphoma
Ibritumomab-tiuxetean action
cytotoxic radiation
Yttrium 90 decays by emission of Beta particles (half life 64hrs)
ADCC and CDC
Avastin target
inhibit vascular endothelial growth factor A
Avastin action
inhibit angiogenesis - withdrawn
IgE binding site
FcER
IgE action
inhibit new IgE production targetting IgE switched cells
Anti-IgE antibody example and target
rhuMAB-E25 humanised (Omalizumab)
bind free IgE
rhuMAB-E25 is non-anaplylactogenic
Omalizumab/Xolair
unable to induce cross-linking and mediator release (mast cells) by masking epitope
rhuMAB-E25 effect and use
IgE unresponsiveness - clonal elimination
allergic rhinitis and allergic asthma
Xolair (future) effects (3)
Omalizumab
reduce exacerbations
reduces need for inhaled corticosteroids
improves asthma and lung functions
Omalizumab target patients
moderate to severe persistent
positive skin test to aeroallergen, not controlled by high corticosteroid
