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Biotechnology > Antibodies > Flashcards

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

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
Q

Detect Ig genes with

A

Southern Blotting

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26
Q

Chimeric antibodies exhibit

A

reduced immunogenecity

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27
Q

Infliximab/Remicade used to treat

A

Inflammatory diseases - crohns and rheumatoid arthritis

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28
Q

Grafting CDR regions from specific rodent monoclonals

A

transfer of specific binding and reduced antigenicity

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29
Q

Framework structure of variable region

A

can help determine shape of CDR loops

designed by crystallography

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30
Q

Omalizumab (Xolair) structure and use

A

humanised, used for severe allergic asthma

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31
Q

Humanised Antibodies development

A

Combine monoclonal murine CDR with human framework

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32
Q

Superhumanised antibodies

A

incorporate human regions into CDR

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33
Q

Fv fragment structure and function

A

bind antigen, heterodimer of VH and VL

unstable as non-covalently linked chains dissociate easily

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34
Q

Single chain fv fragment (scfv)

A

high affinity for antigen and expressed in many hosts

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35
Q

scFv linker

A

domains joined by hydrophilic, flexible peptide - improves expression and folding efficiency
15AA

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36
Q

monomeric scFv

A

compexed into dimers (diabodies) etc.
same specificity - bivalent
different - bispecific - novel functions

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37
Q

Bispecific antibodies construction

A

chemical recombination
cell fusion
genetic manipulation

38
Q

Chemical recombination

A

Fc removed by pepsin digestion
Interchain disulphide removed by reducing agent - 2 mercaptoethanol
fragments purified by chromatography

39
Q

Chemical recombination - rejoining

A

random - oxidative reformation interchain disulphide of hinge
specific by thioester bonding with specific linker

40
Q

BiTE

A

Bispecific T cell engaging antibody

one binds to target epitiope on target cell and other binds effector T cell

41
Q

BiTE blocking

A

block receptors on cell surface

EGFR and insulin-like growth receptor - inhibit tumour growth

42
Q

Trispecific better… (2)

A

tighter binding

enhanced effector function

43
Q

Bispecfic future tech (5)

A 
DART
Modular Ab technology
Bicycle Tech
Dual targeting domains
Chemical generation
44
Q

Single domain antigen binding fragments (dAbs) VhS

A

good affinity

but sticky and difficult to produce in soluble form

45
Q

Heavy chain antibodies (hcAb) IgG

A

no light chain

sharks, camelidae

46
Q

hcAb structure

A

95kDa
single heavy chain variable domain VHH/VNAR
followed immediately by hinge region then CH2/CH3?5xCHNAR

47
Q

hcAb affinity and application

A

similar to Fab and scFv

easier to produce soluble

48
Q

hcAb application (6)

A 
cancer imaging and immunotherapy
cytokine neutralisation
competitive enzyme inhibition
block virus secretion
trypanosome drug delivery
malaria diagnosis
49
Q

Camelisation

A

VL side replaced with VHH-like residues

increase solubility and non-specific binding

50
Q

Plant antibodies

A

Transgenic plants

Ab against dental caries, RA, cholera etc.

51
Q

CaroRX

A

sIgA - dental caries

oral antiseptic then antibody

52
Q

Intrabody

A

Intracellular antibody

expressed within designated cell compartment

53
Q

Intrabody function

A

interruption/modification of target protein functions

scFv, single domain intrabodies intradiabodies (bispecific)

54
Q

Intrabody Use (5)

A 
sequestration protein from compartment 
mediate enzyme function (block/change conformation)
disruption transduction
induction apoptosis
ubiquitin-proteosome degradation
55
Q

Intrabody construction

A

clone V region into vector (retro/adeno)

56
Q

Intrabody treatement (3)

A

Huntingtons
Cancer - alter neoplastic properties
Interruption HIV lifecycle

57
Q

Phage Display

A

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
Q

Phage Display requirements

A

clone gene into filamentous bacteriophage vector with ssDNA and can infect male E.coli (pili)

59
Q

Filamentous bacteriophage attachment

A

N-terminal domain of pIII to tip of the pilus

60
Q

Phage panning

A

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
Q

Uses of phage produced Antibodies

A

ELISA

Soluble antibody fragments

62
Q

Ribosome Display

A

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
Q

Ribosome stalling (2)

A
  • 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
Q

Prokaryotic Ribosome Display - PRM complexes - method

A
  • polysome complex, where complexes were captured with immobilised antibody
  • EDTA released bound mRNA, then —> cDNA
65
Q

Eukaryotic Ribosome Display technique

A

ARM - antibody-ribosome-mRNA complex been developed
captured on antigen coated magnetic beads
mRNA not needed to be dissociated

66
Q

Ribosome display efficient at selecting

A

high affinity scFv

67
Q

Genetically Engineered Mice produce human Ab

A

Denosumab anti-RANKL antibody - treat osteoporosis, bone metastases and induced bone loss

68
Q

TNFalpha in RA

A

formation osteoclasts - bone breakdown
proliferation synoviocytes - joint inflam
expression collagenase by chondrocytes - joint/cartilage destroy

69
Q

anti-TNFalpha effect

A

reduce proinflam cytokines - IL-1,6,8 and GM-CSF

70
Q

Chimeric anti-TNFalpha

A

infliximab/remicade ) with methotrxate

71
Q

IL-1r blockade

A

Anakinra (soluble IL-1Ra)

72
Q

TNFR-Fc fusion protein

A

Etanercept

73
Q

SE RA Antibodies

A

Tuberculosis

74
Q

QUALY

A

Quality-adjusted life year
value for money
no. years of life added - 1.0 = fully healthy year

75
Q

Herceptin target

A

HER-2 - expressed highly in cancer

76
Q

Herceptin action

A

prevents transmission of growth signal to nucleus and stimulated ADCC

77
Q

Rituximab target

A

targets CD20 in Non-Hodgkins Lymphoma

on cancerous B cells

78
Q

Rituximab action

A

stimulates Antibody mediated cellular cytotoxicity

and complement mediated cytotoxicity

79
Q

Gemtuzimab-ozogamacin target

A

CD33 in acute myeloid leukaemia on leukaemic cells

80
Q

Gemtuzimab-ozogamacin action

A

bind CD33 and internalised ozogamacin released
into lysosomes of cells
binds DNA and strand breaks and apoptosis

81
Q

Ibritumomab-tiuxetetan target (IgG1 90Y conjugate)

A

bind CD20 on non-hodgkins lymphoma

82
Q

Ibritumomab-tiuxetean action

A

cytotoxic radiation
Yttrium 90 decays by emission of Beta particles (half life 64hrs)
ADCC and CDC

83
Q

Avastin target

A

inhibit vascular endothelial growth factor A

84
Q

Avastin action

A

inhibit angiogenesis - withdrawn

85
Q

IgE binding site

A

FcER

86
Q

IgE action

A

inhibit new IgE production targetting IgE switched cells

87
Q

Anti-IgE antibody example and target

A

rhuMAB-E25 humanised (Omalizumab)

bind free IgE

88
Q

rhuMAB-E25 is non-anaplylactogenic

Omalizumab/Xolair

A

unable to induce cross-linking and mediator release (mast cells) by masking epitope

89
Q

rhuMAB-E25 effect and use

A

IgE unresponsiveness - clonal elimination

allergic rhinitis and allergic asthma

90
Q

Xolair (future) effects (3)

Omalizumab

A

reduce exacerbations
reduces need for inhaled corticosteroids
improves asthma and lung functions

91
Q

Omalizumab target patients

A

moderate to severe persistent

positive skin test to aeroallergen, not controlled by high corticosteroid

Biotechnology (7 decks)

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