biotechnology insulin ( part2) Flashcards
how is the immune system divided up?
innate immunity- epithelial barriers, soluble mediators, cellular mediators, antimicrobial peptides
adaptive immunity- humoral immunity, cellular immunity
what happens in the immune system during an antbody response?
- Iden5fy-the-dangerous-cell/foreign-par5cle-
- Generate-an5bodies-to-the-exposed-surface-
structures-of-the-cell/par5cle- - Select-the-“best”-an5body-and-then-induce-
clonal-lymphocyte-expansion- - Target-the-cell/par5cle-and-destroy-it-
how many types of immunoglobulins are there?
5 IgA d e g m
what immunoglobulin are monoclonal antibodies usually?
usuallt IgG molesules or modified IgG molecules
what is the structure of an immunoglobulin?
basic structure if 4 subunits:
2 x identical 23-kD light chains (L)
2 x identical 53- to 75-kD heavy chains (H)
what are the associations between the subunits of an immunoglobulin structure?
These subunits associate via disulfide bonds
as well as by non-covalent interactions to
form a Y-shaped symmetric dimer, (L–H)2
Immunoglobulins are glycoproteins - each
heavy chain has an N-linked oligosaccharide
what are Fab fragments?
The Fab fragments (arms of the Y- shaped IgG molecule) consist
of an entire L chain and the N-terminal half of an H chain and
contain IgG’s antigen-binding sites
why is the Fc fragment on the immunoglobulin structure named as such?
The Fc fragment (so named because it is readily
crystalilized) derives from the stem of the Y and
consists of the identical C-terminal segments of
two H chains
what is the job of the FC fragment?
Fc fragments contain the effector sites that
mediate the functions common to a particular
immunoglobulin isotype such as
• Inducing phagocytosis,
• Triggering the complement system
• Directing the transport of Ig molecules to their
sites of action
The Fc region binds to Fc receptors that are
displayed on the surfaces of many types of
immune system cells.
how does the immune system generate antibodies?
The immune system has the capacity to generate antibodies against almost any
antigen that it encounters; it can produce a virtually unlimited variety of
antigen-binding sites.
why is there enormous diversity in antibodies?
One might reasonably expect that immunoglobulin gene expression resembles
that of other proteins in that every distinct H and L chain is encoded by a
separate germline gene.
If this were true, then to encode the billions of different antibodies each
vertebrate appears capable of producing would require huge numbers of these
genes.
what are the two other models for the origin of antibody diversity which have been seriously considered?
- Somatic recombination
Antibody diversity is generated by genetic recombinantion among
relatively few gene segments encoding the variable regions
This process occurs by intra-chromosomal recombination during B cells
differentiation activated by an antigen - Somatic hypermutation
Antibody diversity is generated through a very high proportion of
immunoglobulin gene mutation (hypermutation) during B cell
differentiation
what are monoclonal antibodies and what are they generated from?
A monoclonal an?body (mAb) is a mono-specific an?body i.e. it recognises
and binds just one epitope
Monoclonal an?bodies are generated from a single clonal lymphocyte
why is it not possible to generate homogeneous immunoglobulins in large quantites by cloning a lymphocyte and harvesting the immunoglibulin the clone produced?
as lymphocytes do not grow continuously in culture
what is hybridoma generation?
Hybridomagenera-on allowed virtually unlimited quan//es of a specific mAb
to be generated
Hybridomas are generated by fusing myeloma cells with lymphocytes raised
against that an@gen (that is, isolated from an animal that has been immunized
with the an@gen).
what is the procedure when generating monoclonal antibodies?
- Inject a mouse with an antigen of interest
- Isolate the lymphocytes from the mouse spleen
- Fuse the lymphocytes with mutant mouse myeloma cells
(immortal cells) - Culture the fused cells (Hybridomas) in HAT media:
H: Hypoxanthine
A: Amethopterin (methotrexate, an antifolate)
T: Thymine - The growing hybridomas are then isolated and grown in separate
wells to generate clones - The clones are then screened for the production of the desired
antibody - The gene of interest can be isolated and re-cloned
will all clones generate the required antibody?
no
what is transgenetic technologies?
The term transgenic animal refers to an animal in
which there has been a deliberate modification of
the genome
Foreign DNA is introduced into the animal, using
recombinant DNA technology,
These genes must be transmitted through the germ
line so that every cell, including germ cells, of the
animal contain the same modified genetic material
what is the procedure for transgenic tehcnologies?
- Collection of Fertilized Embryos from Super-ovulated
female mice – ES cell isolation and engineering to KO
mouse IgG genes - Microinjection of DNA (HAC: Human artificial
Chromosome) into engineered ES cell and transfer to
blastocyst - Transgenic births – Chimeric mice
- Analysis of Tail DNA for Transgene
- Mating Transgenic Mice to get full human IgG
repertoire
what will you need no matter what method you use for antibody generation?
No matter what method you use – you will want/need the DNA sequence that encodes
your mAb = because that’s how you make large amounts of it very reproducibly
what is SCID?
SCID = Severe combined immunodeficient (affecting both B and T cell mediated
responses)
what are scid mice used for?
SCID mice are routinely used as model organisms for research into the basic
biology of the immune system, cell transplantation strategies, and the effects of
disease on mammalian systems.
They have been extensively used as hosts for normal and malignant tissue
transplants
what therapeutics are based upon antibody fragments
• Domain andbodies: These are single-domain andbody fragments
• Fab fragments: andgen binding fragment (can be two Fab fragments –
bispecific)
• scFv: single chain variable fragment
• Fc-fusions: Fusion between an immunoglobulin Fc domain and
another pepdde
what is Siha or SiHi cell line?
Human cervical cancer cell line
Easily transfected to generate protein
Epithelial cell line – can be induced to be grown in suspension
what is a purification strategy?
Two main affinity resins are used for the
primary capture chromatography step
how does the chromatography resin work?
primary capture chromatography- removal of main impurities
intermediate chromatography- removal of minor impurities
polishing chromatography: separation of product- related impirities and different isoforms
how are antibodies used to purify?
A single peak on the capture chromatography step does not mean that you only have a single protein species in your eluate Your second chromatography step needs to purify to homogeneity Dimers and aggregates are the biggest form of product-related impurity
what quality attributes would you consider?
functional- effector functions: complement interaction Fc receptor interaction
physiochemical characteristics:
1-N-terminal heterogeneity- pyroglutmate formulation other modifications
-AA modifications- demidation, oxidation,glycation
3-fragmentation- change in hinge region, asp-pro
4-oligosaccharides- fucossylation, sialyation, galactosylation
5-disulfide bonds- free thiols, disulfide shuffling, thioether
6-c-terminal hetrogeneity- lysine processing, priline amidation
what are mAbs?
glycoproteins
what are the glycosylation patterns?
O-linked - OH group of serine and threonine residues
N-linked: addition of Glu3-Man9-GlcNAc2 to NH2-group of asparagine in sequence Asn-X-Ser/Thr in ER
This is subsequently trimmed in the Enodplasmic Reticulum to Man8GlcNAc2
what are some problems with glycosylation patterns?
- Non-human glycosylation patterns
- Hyperglycosylation = high mannose subunit composition
- Yeast will probably glycosylate proteins that are not usually glycosylated in human cells
- Glycosylation is variable = heterogeneous product
- Immunogenicity
