antibody Flashcards
what i’m supposed to know
- Antibody structure, function, basic concepts,
and formats - Antibodies as drugs
- Antibody development strategies and
technologies
what are antibody
are molecules that are produced by B cells for immune response
what are the parts of antibody
Fab: which binds the antigen is consist of heavy and light chain and is region of antigen interaction
Fc: region which identify antibody isotype and recruit effector function like immune cells binding and Fc receptor region interaction.
what is polyclonal antibodies
are mixture of antibodies that are found in the blood serum
what are the characteristic of polyclonal Ab
- they recognize different epitopes of the target/ antigen
-Ab recognize the Ag with different binding strength/ affinity
-bind different epitope with different functionality
they have different affinity because they bind/recognize different epitope
what is monoclonal antibody
is a single antibody that recognize one epitope with defined affinity
what is the target based discovery approach to define an appropriate antigen to target in therapeutic application
- identify the target
-antibody selection on the target
-antibody screening for binding
-screening for function
-in vivo assessment
function based discovery
- antibody selection
-screening for binding
-screening for functionality
-target ID
-in vivo testing
this is the best for identifying the better accessible target for Ab
what is epitope
is a part of antigen recognized by the Ab
what happens when Different antibodies may recognise very similar epitopes
- they compete for binding
- or they can bind to nearby region which allow them to interact with same region of Ag
what is overlapping and non-overlapping epitope
- overlapping epitope is a region of the antigen where 2 or more epitope share the same structural features or share common set of aa. where Ab compete for binding as shown above.
-non-overlapping epitope is when Ag has separate region for Ab binding
what id the characteristic of overlapping epitope
-there is direct competition btn the Ab due to the binding for the same Ag epitope.
- Ab can’t bind simultaneous because of the steric hindrance.
-are found in small antigens
example: Humira interfere with the binding of the TNF to its receptor. by blocking the TNF from binding to its receptor
what is the characteristic of non-overlapping epitope
- multiple antibodies binds to non-overlapped epitopes on the same antigen without steric hindrance
- the epitopes are structural independent
- so, no competitive
-are commonly found in large antigens
what is the importance of high affinity
high affinity interaction are beneficial to develop high functionality.
- longer/ slow dissociation time is desired for high binding affinity
what steps to follow during antibody/antibody fragment development
- primary screening: ELISA,…
-sequencing: NG sequence, Sanger
-Affinity determination: SPR - specificity screening assay; needed for off target interaction
-orthogonal screening: to confirm the previous finding on the interaction test
what is the best method for primary screening
- ELISA- work by staining against the Ab to see how much binding occurs
- flow cytometry screening
- next generation screening
how can you measure the affinity
use SPR
why is important to do cross reactivity test
help us to see if the antibody is targeting what it was supposed to target or if its reacting with other protein/cross-reacting
why is cross activity test required
FOR: -functionality validation
- assessment of side effect: on and off target toxicity
how can i measure epitope specificity
ELISA-epitope mapping assay
,SPR
why is it importance to understand epitope specificity
because it helps us to understand antibody mechanism
what are the Antibody mechanisms of action
- blocking
-signalling
-payload delivery
-induction of apoptosis
-cell killing through car t cells
-checkpoint inhibitor which allow immune control to be active
what are nano bodies
they are Ab like small molecule.
they have short half-live and better tissue penetration
targeting HER2+ breast cancer with antibody based therapeutics
what is the difference between normal and abnormal breast cancer cells
NBCC: has small amount of HER2+ receptor which send signal to cells to divide
ABCC: has large amount of HER2+ which send a lot of signal causing cells to divide and grow quickly
how does anti-HER2 targeted Ab therapy works
- inhibiting dimerization which is weakening the growth signal of cells from HER2+
- receptor internalization: here the Ab promote internalization of HER2+ receptor by engulfing the receptor into the cells via endocytosis. this will reduce signalling and cause HER2+ degradation in lysosome which reduce their activities.
- engagement of ADCs- Ab receptor binds to Ag receptor and those Ab FC region interact with immune cells TC region and they release perforins or payloads that induce apoptotic of cells by directly killing cancer cells and reducing growth signalling
what does Combining antibodies (pertuzumab and trastuzumab) benefit us in Ab therapeutic
by combining Ab it provide more Ag blockage, in this case it will provide more HER2 blockage
explain trastuzmab ADCs
Ab binds to the HER2,
-recruit effector cells via FC region
-binding of FR to Fc lead to release of perforin which leads to attach of HER carrying cells
what is antibody drug conjugation
is an antibody that contain payload and is targeted delivery of high cytotoxic agents.
it directly inhibit downstream signal
explain ADCs assay
-ADC binds to the antigen
-internalization of ADCs through endocytosis
- degradation of ADCs in lysosomes
-leads to the release of payload and drug action
- apoptosis of target cell
benefit of using ADCs
-high affinity
-reduce immunogenicity ( to reduce the formation of anti-drug antibody)
-have long half-life
what is the benefit of ADCs over chemotherapy
efficiency and specific drug delivery to antigen expressing tumour cells
describe ADCs components
- Ab: high affinity, long half-life, decrease immunogenicity
- Linker: stable in circulation, avoid premature release of payload, efficient release of payload
- Payload: able to kill the target cells, high potent agent (IC50), has cytotoxic agent, optimal DAR
- Why are monoclonal antibodies for neuro-oncology difficult to develop? What can be done to improve antibody trials in this area?
- How can other new technologies (CRISPR, CAR-T cells, Cancer mRNA vaccines etc.) can be used in combination or in conjunction with monoclonal antibodies?
- What other diseases could monoclonal antibodies would be beneficial in treatment? What would be the mechanism of action in those settings?
1.
a) why:
- to cross BBB is very challenging
- tumor heterogeneity
-immunosuppression microenvironment
b) improve:
- enhance BBB permeability
- combine therapy: by combining Mab with checkpoint inhibitor
- better target selection
2.
a) CRISPR:
- Mab optimization: gene editing can improve the design of Mab leading to increase in binding affinity
- target gene editing: can be used to knockout immunosuppressive gene in tumor making it easy for Mab
b) CAR-T cells:
- can be used to target negative tumour cells while Mab is targeting positive tumour cells
- enhance tumour microenvironment: by releasing cytokines that improve Mab activity
c)mRNA Vaccines;
- it enhance immune system recognition of cancer cells
- Autoimmune Diseases: using checkpoints
- COVID-19 or HIV: Mab can block viral entry into the host cells
