antibody

Question 1

what i’m supposed to know

Answer 1

• Antibody structure, function, basic concepts,
  and formats
• Antibodies as drugs
• Antibody development strategies and
  technologies

Question 2

what are antibody

Answer 2

are molecules that are produced by B cells for immune response

Question 3

what are the parts of antibody

Answer 3

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.

Question 4

what is polyclonal antibodies

Answer 4

are mixture of antibodies that are found in the blood serum

Question 5

what are the characteristic of polyclonal Ab

Answer 5

• 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

Question 6

what is monoclonal antibody

Answer 6

is a single antibody that recognize one epitope with defined affinity

Question 7

what is the target based discovery approach to define an appropriate antigen to target in therapeutic application

Answer 7

• identify the target
  -antibody selection on the target
  -antibody screening for binding
  -screening for function
  -in vivo assessment

Question 8

function based discovery

Answer 8

• 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

Question 9

what is epitope

Answer 9

is a part of antigen recognized by the Ab

Question 10

what happens when Different antibodies may recognise very similar epitopes

Answer 10

• they compete for binding
• or they can bind to nearby region which allow them to interact with same region of Ag

Question 11

what is overlapping and non-overlapping epitope

Answer 11

• 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

Question 12

what id the characteristic of overlapping epitope

Answer 12

-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

Question 13

what is the characteristic of non-overlapping epitope

Answer 13

• 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

Question 14

what is the importance of high affinity

Answer 14

high affinity interaction are beneficial to develop high functionality.
- longer/ slow dissociation time is desired for high binding affinity

Question 15

what steps to follow during antibody/antibody fragment development

Answer 15

• 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

Question 16

what is the best method for primary screening

Answer 16

• ELISA- work by staining against the Ab to see how much binding occurs
• flow cytometry screening
• next generation screening

Question 17

how can you measure the affinity

Answer 17

use SPR

Question 18

why is important to do cross reactivity test

Answer 18

help us to see if the antibody is targeting what it was supposed to target or if its reacting with other protein/cross-reacting

Question 19

why is cross activity test required

Answer 19

FOR: -functionality validation
- assessment of side effect: on and off target toxicity

Question 20

how can i measure epitope specificity

Answer 20

ELISA-epitope mapping assay
,SPR

Question 21

why is it importance to understand epitope specificity

Answer 21

because it helps us to understand antibody mechanism

Question 22

what are the Antibody mechanisms of action

Answer 22

• blocking
  -signalling
  -payload delivery
  -induction of apoptosis
  -cell killing through car t cells
  -checkpoint inhibitor which allow immune control to be active

Question 23

what is checkpoint inhibitor

Answer 23

when checkpoint protein of T cells recognize and binds to their partner from tumor cells they sent off signal which prevent T cells from targeting tumor cells.
so, by using checkpoint inhibitor it binds to checkpoint proteins and prevent the binding which prevent sent of off signal and allow T cells to kill/ target tumor cells.

Question 24

what are nano bodies

Answer 24

they are Ab like small molecule.
they have short half-live and better tissue penetration

Question 25

targeting HER2+ breast cancer with antibody based therapeutics

Question 26

what is the difference between normal and abnormal breast cancer cells

Answer 26

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

Question 27

how does anti-HER2 targeted Ab therapy works

Answer 27

- 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

Question 28

what does Combining antibodies (pertuzumab and trastuzumab) benefit us in Ab therapeutic

Answer 28

by combining Ab it provide more Ag blockage, in this case it will provide more HER2 blockage

Question 29

explain trastuzmab ADCs

Answer 29

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

Question 30

what is antibody drug conjugation

Answer 30

is an antibody that contain payload and is targeted delivery of high cytotoxic agents. it directly inhibit downstream signal

Question 31

explain ADCs assay

Answer 31

-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

Question 32

benefit of using ADCs

Answer 32

-high affinity -reduce immunogenicity ( to reduce the formation of anti-drug antibody) -have long half-life

Question 33

what is the benefit of ADCs over chemotherapy

Answer 33

efficiency and specific drug delivery to antigen expressing tumour cells

Question 34

describe ADCs components

Answer 34

- 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

Question 35

1. Why are monoclonal antibodies for neuro-oncology difficult to develop? What can be done to improve antibody trials in this area? 2. How can other new technologies (CRISPR, CAR-T cells, Cancer mRNA vaccines etc.) can be used in combination or in conjunction with monoclonal antibodies? 2. What other diseases could monoclonal antibodies would be beneficial in treatment? What would be the mechanism of action in those settings?

Answer 35

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 -Cross link them with effector cells to increase apoptosis - better target selection - humanized Ab 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 3. - Autoimmune Diseases: using checkpoints - COVID-19 or HIV: Mab can block viral entry into the host cells