biotechnology insulin ( part2)

Question 1

how is the immune system divided up?

Answer 1

innate immunity- epithelial barriers, soluble mediators, cellular mediators, antimicrobial peptides

adaptive immunity- humoral immunity, cellular immunity

Question 2

what happens in the immune system during an antbody response?

Answer 2

1. Iden5fy-the-dangerous-cell/foreign-par5cle-
2. Generate-an5bodies-to-the-exposed-surface-
   structures-of-the-cell/par5cle-
3. Select-the-“best”-an5body-and-then-induce-
   clonal-lymphocyte-expansion-
4. Target-the-cell/par5cle-and-destroy-it-

Question 3

how many types of immunoglobulins are there?

Answer 3

5
IgA
d
e
g
m

Question 4

what immunoglobulin are monoclonal antibodies usually?

Answer 4

usuallt IgG molesules or modified IgG molecules

Question 5

what is the structure of an immunoglobulin?

Answer 5

basic structure if 4 subunits:
2 x identical 23-kD light chains (L)
2 x identical 53- to 75-kD heavy chains (H)

Question 6

what are the associations between the subunits of an immunoglobulin structure?

Answer 6

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

Question 7

what are Fab fragments?

Answer 7

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

Question 8

why is the Fc fragment on the immunoglobulin structure named as such?

Answer 8

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

Question 9

what is the job of the FC fragment?

Answer 9

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.

Question 10

how does the immune system generate antibodies?

Answer 10

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.

Question 11

why is there enormous diversity in antibodies?

Answer 11

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.

Question 12

what are the two other models for the origin of antibody diversity which have been seriously considered?

Answer 12

1. 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
2. Somatic hypermutation
   Antibody diversity is generated through a very high proportion of
   immunoglobulin gene mutation (hypermutation) during B cell
   differentiation

Question 13

what are monoclonal antibodies and what are they generated from?

Answer 13

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

Question 14

why is it not possible to generate homogeneous immunoglobulins in large quantites by cloning a lymphocyte and harvesting the immunoglibulin the clone produced?

Answer 14

as lymphocytes do not grow continuously in culture

Question 15

what is hybridoma generation?

Answer 15

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).

Question 16

what is the procedure when generating monoclonal antibodies?

Answer 16

1. Inject a mouse with an antigen of interest
2. Isolate the lymphocytes from the mouse spleen
3. Fuse the lymphocytes with mutant mouse myeloma cells
   (immortal cells)
4. Culture the fused cells (Hybridomas) in HAT media:
   H: Hypoxanthine
   A: Amethopterin (methotrexate, an antifolate)
   T: Thymine
5. The growing hybridomas are then isolated and grown in separate
   wells to generate clones
6. The clones are then screened for the production of the desired
   antibody
7. The gene of interest can be isolated and re-cloned

Question 17

will all clones generate the required antibody?

Answer 17

no

Question 18

what is transgenetic technologies?

Answer 18

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

Question 19

what is the procedure for transgenic tehcnologies?

Answer 19

1. Collection of Fertilized Embryos from Super-ovulated
   female mice – ES cell isolation and engineering to KO
   mouse IgG genes
2. Microinjection of DNA (HAC: Human artificial
   Chromosome) into engineered ES cell and transfer to
   blastocyst
3. Transgenic births – Chimeric mice
4. Analysis of Tail DNA for Transgene
5. Mating Transgenic Mice to get full human IgG
   repertoire

Question 20

what will you need no matter what method you use for antibody generation?

Answer 20

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

Question 21

what is SCID?

Answer 21

SCID = Severe combined immunodeficient (affecting both B and T cell mediated
responses)

Question 22

what are scid mice used for?

Answer 22

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

Question 23

what therapeutics are based upon antibody fragments

Answer 23

• 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

Question 24

what is Siha or SiHi cell line?

Answer 24

Human cervical cancer cell line
Easily transfected to generate protein
Epithelial cell line – can be induced to be grown in suspension

Question 25

what is a purification strategy?

Answer 25

Two main affinity resins are used for the

primary capture chromatography step

Question 26

how does the chromatography resin work?

Answer 26

primary capture chromatography- removal of main impurities
intermediate chromatography- removal of minor impurities
polishing chromatography: separation of product- related impirities and different isoforms

Question 27

how are antibodies used to purify?

Answer 27

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

Question 28

what quality attributes would you consider?

Answer 28

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

Question 29

what are mAbs?

Answer 29

glycoproteins

Question 30

what are the glycosylation patterns?

Answer 30

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

Question 31

what are some problems with glycosylation patterns?

Answer 31

• 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