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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 (91 cards)

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1
Q

Ab Functions

A

Bind specifically to molecules on pathogen/antigen that created immune response
recruit cells and molecules to destroy pathogen/antigen once bound

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2
Q

Structure

A

each chain series of similar sequences, each repeat corresponds to domain

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3
Q

CH2 domains not interact due to

A

carbohydrate side chains

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4
Q

Domain structure

A

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

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5
Q

The most variable regions and structure

A

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

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6
Q

Regions between HV regions -

A

framework regions FR1,2,3+4

form Beta sheets

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7
Q

Polyclonal antibody

A

different antibodies even against same antigen

from immunised animal - immunoglobulin isolated

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8
Q

monoclonal antibody

A

uniform population

same binding specificity, affinity and biological activity, produced by identical hybridoma cells

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9
Q

Hybridoma cells

A

fusion of immune lymphocytes with myeloma cells - antibody secreting and long term survival potential

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10
Q

Monoclonal antibodies - Immunisation protocol

A

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

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11
Q

Myeloma cell properties required

A

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)

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12
Q

Fusion protocol

A

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

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13
Q

Nucleic acid synthesis routes

A

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

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14
Q

HAT medium contains

A

aminopterin - antibiotic blocks de novo
hypoxanthine and thymidine - for salvage
enzymes not present in myleoma cells

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15
Q

Only hybrid survive

A

B cells have finite lifespan

myeloma cells have no HGPRT

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16
Q

Screening Test

A

ELISA - as similar to final assay

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17
Q

Cloning protocol

A

Limited Dilution
supplemented with feeder cells (peritonal macrophages, splenocytes) provide growth factors
supernatant tested for antibodies

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18
Q

Crypreservation

A

store in liquid nitrogen

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19
Q

Polyclonal antibody qualities (6)

A 
cross react
limited supply
choice of species
precipitate
best with pure antigen
easy to screen
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20
Q

Monoclonal antibody qualities (6)

A 
specific
unlimited
limited species (rat, mouse)
not precipitate
not need purified antigen
need good screening
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21
Q

HAMA

A

Human anti mouse antibody

mouse antibodies only 60-70% similar

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22
Q

Possibly prevent by

A

replacing non-specific part with human part

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23
Q

Human antigen binding fragments created from

A

combinational libraries as bacteria express Fab and Fvs

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24
Q

Ab ideal for recombination as

A

each domain is coded for by different exon - chimeric

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25
Detect Ig genes with
Southern Blotting
26
Chimeric antibodies exhibit
reduced immunogenecity
27
Infliximab/Remicade used to treat
Inflammatory diseases - crohns and rheumatoid arthritis
28
Grafting CDR regions from specific rodent monoclonals
transfer of specific binding and reduced antigenicity
29
Framework structure of variable region
can help determine shape of CDR loops | designed by crystallography
30
Omalizumab (Xolair) structure and use
humanised, used for severe allergic asthma
31
Humanised Antibodies development
Combine monoclonal murine CDR with human framework
32
Superhumanised antibodies
incorporate human regions into CDR
33
Fv fragment structure and function
bind antigen, heterodimer of VH and VL | unstable as non-covalently linked chains dissociate easily
34
Single chain fv fragment (scfv)
high affinity for antigen and expressed in many hosts
35
scFv linker
domains joined by hydrophilic, flexible peptide - improves expression and folding efficiency 15AA
36
monomeric scFv
compexed into dimers (diabodies) etc. same specificity - bivalent different - bispecific - novel functions
37
Bispecific antibodies construction
chemical recombination cell fusion genetic manipulation
38
Chemical recombination
Fc removed by pepsin digestion Interchain disulphide removed by reducing agent - 2 mercaptoethanol fragments purified by chromatography
39
Chemical recombination - rejoining
random - oxidative reformation interchain disulphide of hinge specific by thioester bonding with specific linker
40
BiTE
Bispecific T cell engaging antibody | one binds to target epitiope on target cell and other binds effector T cell
41
BiTE blocking
block receptors on cell surface | EGFR and insulin-like growth receptor - inhibit tumour growth
42
Trispecific better... (2)
tighter binding | enhanced effector function
43
Bispecfic future tech (5)
``` DART Modular Ab technology Bicycle Tech Dual targeting domains Chemical generation ```
44
Single domain antigen binding fragments (dAbs) VhS
good affinity | but sticky and difficult to produce in soluble form
45
Heavy chain antibodies (hcAb) IgG
no light chain | sharks, camelidae
46
hcAb structure
95kDa single heavy chain variable domain VHH/VNAR followed immediately by hinge region then CH2/CH3?5xCHNAR
47
hcAb affinity and application
similar to Fab and scFv | easier to produce soluble
48
hcAb application (6)
``` cancer imaging and immunotherapy cytokine neutralisation competitive enzyme inhibition block virus secretion trypanosome drug delivery malaria diagnosis ```
49
Camelisation
VL side replaced with VHH-like residues | increase solubility and non-specific binding
50
Plant antibodies
Transgenic plants | Ab against dental caries, RA, cholera etc.
51
CaroRX
sIgA - dental caries | oral antiseptic then antibody
52
Intrabody
Intracellular antibody | expressed within designated cell compartment
53
Intrabody function
interruption/modification of target protein functions | scFv, single domain intrabodies intradiabodies (bispecific)
54
Intrabody Use (5)
``` sequestration protein from compartment mediate enzyme function (block/change conformation) disruption transduction induction apoptosis ubiquitin-proteosome degradation ```
55
Intrabody construction
clone V region into vector (retro/adeno)
56
Intrabody treatement (3)
Huntingtons Cancer - alter neoplastic properties Interruption HIV lifecycle
57
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
58
Phage Display requirements
clone gene into filamentous bacteriophage vector with ssDNA and can infect male E.coli (pili)
59
Filamentous bacteriophage attachment
N-terminal domain of pIII to tip of the pilus
60
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
61
Uses of phage produced Antibodies
ELISA | Soluble antibody fragments
62
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
63
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
64
Prokaryotic Ribosome Display - PRM complexes - method
- polysome complex, where complexes were captured with immobilised antibody - EDTA released bound mRNA, then ---> cDNA
65
Eukaryotic Ribosome Display technique
ARM - antibody-ribosome-mRNA complex been developed captured on antigen coated magnetic beads mRNA not needed to be dissociated
66
Ribosome display efficient at selecting
high affinity scFv
67
Genetically Engineered Mice produce human Ab
Denosumab anti-RANKL antibody - treat osteoporosis, bone metastases and induced bone loss
68
TNFalpha in RA
formation osteoclasts - bone breakdown proliferation synoviocytes - joint inflam expression collagenase by chondrocytes - joint/cartilage destroy
69
anti-TNFalpha effect
reduce proinflam cytokines - IL-1,6,8 and GM-CSF
70
Chimeric anti-TNFalpha
infliximab/remicade ) with methotrxate
71
IL-1r blockade
Anakinra (soluble IL-1Ra)
72
TNFR-Fc fusion protein
Etanercept
73
SE RA Antibodies
Tuberculosis
74
QUALY
Quality-adjusted life year value for money no. years of life added - 1.0 = fully healthy year
75
Herceptin target
HER-2 - expressed highly in cancer
76
Herceptin action
prevents transmission of growth signal to nucleus and stimulated ADCC
77
Rituximab target
targets CD20 in Non-Hodgkins Lymphoma | on cancerous B cells
78
Rituximab action
stimulates Antibody mediated cellular cytotoxicity | and complement mediated cytotoxicity
79
Gemtuzimab-ozogamacin target
CD33 in acute myeloid leukaemia on leukaemic cells
80
Gemtuzimab-ozogamacin action
bind CD33 and internalised ozogamacin released into lysosomes of cells binds DNA and strand breaks and apoptosis
81
Ibritumomab-tiuxetetan target (IgG1 90Y conjugate)
bind CD20 on non-hodgkins lymphoma
82
Ibritumomab-tiuxetean action
cytotoxic radiation Yttrium 90 decays by emission of Beta particles (half life 64hrs) ADCC and CDC
83
Avastin target
inhibit vascular endothelial growth factor A
84
Avastin action
inhibit angiogenesis - withdrawn
85
IgE binding site
FcER
86
IgE action
inhibit new IgE production targetting IgE switched cells
87
Anti-IgE antibody example and target
rhuMAB-E25 humanised (Omalizumab) | bind free IgE
88
rhuMAB-E25 is non-anaplylactogenic | Omalizumab/Xolair
unable to induce cross-linking and mediator release (mast cells) by masking epitope
89
rhuMAB-E25 effect and use
IgE unresponsiveness - clonal elimination | allergic rhinitis and allergic asthma
90
Xolair (future) effects (3) | Omalizumab
reduce exacerbations reduces need for inhaled corticosteroids improves asthma and lung functions
91
Omalizumab target patients
moderate to severe persistent | positive skin test to aeroallergen, not controlled by high corticosteroid

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