Lecture 6 - Antibodies

Question 1

definition of an immunoglobulin

Answer 1

protein used by immune system to neutralize foreign objects like bacteria and viruses

Question 2

describe the structure of antibodies

Answer 2

heavy and light chain

heavy = 2 chains of constant effector region at bottom, 1 chain with Ag-binding site on each arm of the Y

light = 1 chain with Ag-binding site on each arm of the Y

Question 3

what happens if we engineer an Ab to lose the Fc portion and keep Ag-binding region?

Answer 3

receptor-Ab interaction without activating immune system

Question 4

what are the complementarity-determining regions?

Answer 4

part of the variable chains where the Ab binds to the Ag

Question 5

what is a paratope?

Answer 5

a set of CDRs

Question 6

CDRs are the most _______ part of Ab, therefore:

Answer 6

CDRs are the most variable part of the Ab, therefore they are crucial to the diversity of Ab

Question 7

how many CDRs are there on an immunoglobulin?

Answer 7

each variable domain has 3 CDRs (CDR1/2/3) and sicne there are 2 variable domains, an Ab has 6 CDRs

Question 8

how many CDRs are there on an IgM?

Answer 8

60 CDRs

Question 9

how can we use CDRs in drug development?

Answer 9

modify CDRs to have specificity for certain epitope

Question 10

what are polyclonal Ab?

what are they made by?

how do they bind Ag?

Answer 10

mixture of heterogenous Ab

produced by diff B cell clones in the body

can recognize and bind to many epitopes of a single antigen (many CDRs involved!)

Question 11

what are monoclonal Ab?

what are they made by?

how do they bind Ag?

Answer 11

one type of Ab

produced by identical B cells that are clones from a single parent cell

can only recognize one epitope of an antigen

Question 12

how are antibodies involved in the complement system?

Answer 12

Fc portion can bind complement proteins and help the immune cell recognize pathogen thru complement cascade

Question 13

how are antibodies involved in macrophages?

Answer 13

Ab binds Fc receptor on macrophages –> Ab bind pathogen and opsonize it so the macrophage can phagocytose it

Question 14

describe Ab in RSV-infected cell

Answer 14

cell infected with RSV expresses specific protein on its surface –> allows for production of Ab that binds the protein –> NK cell recognizes the Fc and kill the infected cell

Question 15

what is the scFv fragment?

Answer 15

made of the variable domain of the heavy and light chains of a mAb (i.e. the Ag-binding part)

when we chop them up for therapeutic purposes, we express as 1 polypeptide chain with a linker btwn the chains

Question 16

what is the Fab fragment?

Answer 16

variable domain of each chain and the first constant region (the entire upper part of the Y)

Question 17

how do our bodies make Ab? (3 steps)

Answer 17

1. B cell activated when it meets Ag
2. B cell presents Ag to matching T cell
3. T cell secretes cytokines to help the B cell multiply and mature into Ab-producing plasma cells

Question 18

2 general ways to produce engineered Ab

Answer 18

1. inject Ag into rabbit –> B cells activated and make pAb –> collect antiserum containing pAb
2. inject Ag into mouse –> fuse spleen cells with immortlaized myeloma cells –> B cells can now replicate well and make lots of mouse Ab (can be pAb or mAb)

Question 19

what is phage display?

Answer 19

genetically modify phages so that they can encode variable region that binds epitope –> phages can replicate easily so will make lots Ab (highthroughput!)

test the Ab against Ag –> find the best Ab, put into B cells so they can make the Ab to be used

Question 20

how can we use a transgenic mouse to make human Ab?

Answer 20

humanize mouse to make human B cells and therefore human Ab

Question 21

how can we use human B cells to make Ab?

Answer 21

immortalize a B cell from human and screen for specific Ag

Question 22

describe PSEUDOMONAS AERUGINOSA

Answer 22

• gram negative opportunistic bacteria
• causes pneumonia in ventilated patients –> disrupts lung barrier and causes bacteremia
• multi-drug resistant!

Question 23

what is the role of T3SS in PA?

what happens if it is deleted?

Answer 23

injects mutliple virulence factors into host cells and is used for pore formation

deleted –> bacteria less infective

Question 24

what is PsI exopolysaccharide?

Answer 24

allows for:
- COLONIZATION –> promotes bacterial aggregation and tissue adherence
- PERSISTENCE –> component in biofilm formation/maintenance

also involved in immune evasion

Question 25

describe the 3 Ab used to target PA

Answer 25

all are bispecific Ab that targrets PsI and PcrV portion of T3SS

1. ScFv portion that binds Ag is added next to Fab
2. ScFv portion is added on bottom, i.e. on Fc portion
3. ScFv portion is added btwn Fc and Fab so the variable chain is extended (THEY USED THIS ONE)

Question 26

What happened when they tested anti-PcrV, anti-Psl, and the bispecific Ab for opsonization?

Answer 26

anti-PcrV was poor opsonin but anti-Psl and bispecific Ab were good opsonins

Question 27

efficacy of bispecific Ab in vivo when the Ab was used as prophylaxis

Answer 27

(gave Ab, then infection)

looking at % survival, bispecific Ab was successful only at high concentrations

Question 28

efficacy of bispecific Ab in vivo when the Ab was used as treatment

Answer 28

(gave infection, then Ab)

looking at % survival, bispecific Ab was successful only at high concentrations

Question 29

in vitro vs in vivo results of the anti-PcrV Ab

Answer 29

anti-PcrV Ab had no opsonin effect but did affect survival

therefore, involved in something other than just assisting immune cells and phagocytosis

Question 30

what is gene therapy?

Answer 30

corrects an underlying genetic problem

Question 31

what was the first method of gene therapy?

Answer 31

gene transfer / gene addition

Question 32

2 purposes of gene transfer/gene addition

Answer 32

1. introduce NEW gene into cells to help fight disease
2. introduce NON-FAULTY version of a gene to replace the altered of the gene

Question 33

example of gene therapy in B cells

Answer 33

can inject DNA code that allows our bodies to make Ab from B cells

Question 34

4 ways that gene therapy can help disease

Answer 34

1. fix genetic alteration underlying a disorder
2. turn ON a gene to fight a disease
3. turn OFF a gene that is functioning improperly
4. remove a piece of DNA that is impairing gene function and causing disease

Question 35

development of gene therapy for leukemia (6 steps)

Answer 35

1. took healthy T cells from donor
2. first base edit –> disabled the targeting mechanism of T cells so they would not attack the recipient’s body
3. second base edit –> removed CD7 marker on T cells
4. third base edit –> “invisibility cloak” that prevents cells from being killed by chemotherapy drug
5. fourth modification –> make T cells target anything with CD7 and kill T cells, including cancerous T cells
6. the recipient’s immune system is then rebuilt with bone-marrow transplant

Question 36

what virus do we modify to use as vector for gene therapy? how do we modify it?

Answer 36

ADENOVIRUS –> remove certain replication genes and replace with cassette that expresses foreign therapeutic gene (replication-DEFECTIVE)

Question 37

when are adenovirus vectors used?

Answer 37

for gene therapy as vaccines and for cancer therapy

Question 38

how are adenovirus vectors used for cancer therapy?

Answer 38

they are replication-COMPETENT and can replicate preferentially in cancer cells to destroy them

Question 39

how can bacteriophages be used as therapy?

Answer 39

to treat antimicrobial resistance

Question 40

6 ways antibodies are used to clear infections

Answer 40

1. help body recognize a pathogen
2. block entry of pathogen into a cell
3. neutralize a pathogen’s toxin
4. block secretion of a toxin
5. block protein-protein interaction
6. treatment