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Biomedical technology > Antibodies > Flashcards

Antibodies Flashcards

1
Q

What is Rituximab?

A

an antibody therapy targetting CD20 on b cells, for follicular lymphoma,
used in cancer and autoimmune disease (arthritis, multiple sclerosis).
Highly successful very widely used
Produce by genentech, first mAb developed, a chimeric antibody – first a mouse antibody then constant region replaced by human framework

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2
Q

what is Herceptin?

A
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3
Q

what is Avastin?
Bevacizumab

A

a recombinant humanized monoclonal immunoglobulin antibody that has two antigen-binding domains and blocks all active forms of vascular endothelial growth factor-A.
VEGFR targeting, prevent angiogenesis in cancer, not as successful as hoped, but has application in eye disease, age related macular degeneration, has transformed the quality of life with this

(at the back of the eye there is increased vegf production causing vessel growth that pierces through the retina and causes blindness)

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4
Q

what is humira?

A

human antibody for TNF, phage display using human material, successful

(won nobel prize for the technology used to make this antibody)

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5
Q

what is Nivolumab?

A

targets CTLA4, human antibody, over expressed in tumour stopping immune cells from killing them, targeting prevents survival, nobel prize for the biology behind this

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6
Q

what has been the most sold antibody in 2013 and 2020?

A

humira
$5.2 billion in 2013, $6.6 billion in 2020

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7
Q

give 4 examples of TNFalpha antibody based biologics

A
  • Infliximab (IgG1 Fc & murine Fab, monoclonal antibodies)
  • Adalimumab (IgG1 Fc & human Fab, monocloncal antibodies)
  • Etanercept (recombinant p75 TNF receptor/Fc fusion protein)
  • Certolizumab - Pegol (PEGylated Fab’ fragment/Fc-free
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8
Q

describe the structure of an antibody

A

two light chains and two heavy chains, linked together with covalent (S-S bridges) and non covalent forces.
Antibodies are made up by variable (V) and constant regions (C).
The antigen binding activity is found on the variable region, whereas the Effector function activity is found on the C region

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9
Q

what is VDj recombination?

A

VDJ recombination is the process of somatic recombination by which T cells and B cells randomly assemble different gene segments – known as variable (V), diversity (D) and joining (J) genes – in order to generate unique receptors (known as antigen receptors) that can collectively recognize many different types of molecule

It results in the highly diverse repertoire of antibodies/immunoglobulins and T cell receptors (TCRs) found in B cells and T cells, respectively.

occurs only in developing lymphocytes during the early stages of T and B cell maturation

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10
Q

describe the process from DNA to polypeptide chain of light chain formation

A

In the germline DNA, two segments V and J are brought together and joined
then the gene segement for the constant (C) region is brought in
the DNA is then transcribed
now the primary RNA transcript has a polyA tail added to the constant region, after which splicing occurs to bring the constant regions closer to join VJ
the mRNA is translated and the polyA tail removed leading to the final light chain protein

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11
Q

what are the three gene locus of an antibody?

A

lamda and kappa light chain locus
heavy chain locus

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12
Q

where are the antibody binding sites found on an antibody?

A

within the variable region

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13
Q

where is the effector function activity found in an antibody?

A

within the constant (Fc) region

where interaction with complement proteins and specialized Fc-receptors occurs

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14
Q

the isotype of an antibody is determined by what region of the antibody?

A

the carboxy terminus of the heavy chain
(Fc constant region)

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15
Q

where are the complimentary determining regions in antibodies?

how many are there?

A

There are three CDRs (CDR1, CDR2 and CDR3), arranged non-consecutively, on the amino acid sequence of a variable domain of an antigen receptor.

Since the antigen receptors are typically composed of two variable domains (on two different polypeptide chains, heavy and light chain), there are six CDRs for each antigen receptor that can collectively come into contact with the antigen. A single antibody molecule has two antigen receptors and therefore contains twelve CDRs total. There are three CDR loops per variable domain in antibodies.

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16
Q

how is the diversity of antibodies generated?

A

VDJ recombination

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17
Q

where does recombination occur?
(sequences)

A

at recombination signal sequences RSS (can have different spacers 23 vs 12, and they must be different ones to be combined

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18
Q

describe vdj recombination in the heavy chain

A

gene segments of D and J are brought together and joined
the V segment is them also brought in and joined with DJ
finally the 3 C domains are brought in, tagged with a poly A tail and joined to the VDJ DNA
After splicing the sequence is transcribed
The polyA tail is removed and the mRNA is translated to create the heavy chain protein

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19
Q

give a breif functional differentiation between the subclasses of antibodies

A

Valency (IgM) - important in primary response
Complement activation - IgM, IgG3
Opsonization - IgG1, IgG3
Placental transfer - IgG2, IgG4
Binding to mast cells and basophils - IgE
Secretion onto mucosal surfaces - IgA

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20
Q

how do the subclasses of antibody differ in structure?

A
  • IgG2 has three or four different hinge regions that make the molecule very stable making it more stiff (than IgG1)
  • IgG3 has a very long hinge regions
  • IgG4 looks similar to IgG2
  • IgA is a dimer
  • IgM is a pentamer
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21
Q

what makes IgA a promising antibody for tumours

A

because neutrophils can interact with them and become an effector cells that can contribute to the antitumour effect

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22
Q

describe the principle of vaccination

A

The first response is called a primary response, first apparent several days after encouter with antigen. Re-encounter with the same antigen causes a secondary response and is more rapid and powerful (higher serum antibody and lasting twice as long).
A vaccination triggers a primary response so that when you are infected with the real pathogen then the stronger secondary response can be triggered - immune system doesnt have to learn because there is already memory (B memeory cells)
Through class switching and affinity maturation you can get much stronger and more functional/effective antibodies

vaccination is almost like natural antibody engineering

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23
Q

describe the mechanism of subclass switching

A

In reactivation there is an enzyme called AID that recognises switch sequences in DNA. AID selectively targets the switch regions and nicks them on both strands. this causes looping out of the DNA and switch region recombination to produce a new subclass of antibody with the same antigen specificty

  • Isotype switching ensures fusion of VH to other heavy chain isotype (no change in epitope recognition) Isotype light chain not changed
  • Initiated by Switch (S) sequences and/ catalysed by activation induced cytidine deaminase (AID)
  • Looping out of DNA puts VH in juxtaposition of new isotype
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24
Q

describe the mechanism of subclass switching

A

In reactivation there is an enzyme called AID that recognises switch sequences in DNA. AID selectively targets the switch regions and nicks them on both strands. this causes looping out of the DNA and switch region recombination to produce a new subclass of antibody with the same antigen specificty

  • Isotype switching ensures fusion of VH to other heavy chain isotype (no change in epitope recognition) Isotype light chain not changed
  • Initiated by Switch (S) sequences and/ catalysed by activation induced cytidine deaminase (AID)
  • Looping out of DNA puts VH in juxtaposition of new isotype
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25
Q

what are the functions of antibodies?

A
  • neutralization,(e.g., blocking, interaction with cellular receptor)
  • opsonization, activation complement system leading to lysis pathogen, and ingestion by phagocytes
  • activation NK cells by binding to Fc receptors
  • activation mast cells by interaction with IgE receptor
  • transport across epithelium: secretion into lumen of gut, milk, saliva, sweat and tears to combat parasites, microbes and viruses outside the body
  • transport across placenta to supply fetus with protective antibodies
  • diffusion in extravascular sites of damaged or infected tissues (related to size of antibody)
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26
Q

during optimisation od the immune repsonse most of the changes in the DNA sequence of antibodies occurs in what regions?

A

CDR regions

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27
Q

what antibodies subclasses are best at neutralisation?

A

IgG1
IgG2
IgG3
IgG4
IgA

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28
Q

what antibodies subclasses are best at opsonisation?

A

IgG1
and IgG3 (just a lil less)

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29
Q

what antibodies subclasses are best at sensitisation for killing by NK cells?

A

IgG1 and IgG3

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30
Q

what antibodies subclasses are best at sensitisation of mast cells?

A

IgE

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31
Q

what antibodies subclasses are best at activation of complement system?

A

IgM and IgG3
(IgG1 a lil less)

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32
Q

what antibodies subclasses are best at transport across the epithelium?

A

IgA
(dimer)

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33
Q

what antibodies subclasses are best at transport across the placenta?

A

IgG1
(IgG3 and IgG4 a lil less)

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34
Q

what antibodies subclasses are best at diffusion into extravascular sites?

A

IgG1
IgG2
IgG3
IgG4
(IgA monomer a lil less)

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35
Q

which antibody has the highest mean serum level?

A

IgG1 - 9mg/ml

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36
Q

why are third booster vaccines beneficial?

why?

A
  • Increase in antibody concentrations (plasma)
    – Due to clonal expansion of specific B cells & memory
  • Increase in antibody affinities
    – Mostly seen with IgG as well as IgA, IgE
    – Secreted by switched memory B cells
    – “Affinity maturation” due to somatic hypermutation
    (Occurs in variable region of antibody – small changes that change the specificity of the antibody, only those with better affinity with clonally expand)
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37
Q

what is the difference between somatic hypermutation and class switching?

A

somatic hypermutation occurs in variable region of antibody – small changes that change the specificity of the antibody, only those with better affinity with clonally expand
in class switching the constant-region portion of the antibody heavy chain is changed, but the variable region of the heavy chain stays the same

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38
Q

during optimisation for the immune repsonse where do most of the change occur?

A

in the CDR regions
important for specificity and affinity

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39
Q

generally how long do IgG antibodies circulate?

A

three weeks
(more than other proteins)

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40
Q

how long do IgA or IgM antibodies circulate?

A

6-8 days

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41
Q

why do antibodies have a longer half life that most proteins?

describe the mechanism that allows this

A

due to the recycling mechanism
antibodies are taken up by epithelial cells by non specific binging
into a lysosome (pH 6) where they bind to FcRn (neonatal Fc receptor)
other proteins are degraded but the FcRn saves the antibody and delivered back into the blood due to the change in pH (pH7)

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42
Q

why is the ‘recycling’ of antibodies a pH dependent process?

A

antibody is taken up in lysosome which has low pH(pH6.0), binding to antibody and recycled to surface because the pH in the blood is 7 – this allows it to be recycled not degraded

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43
Q

give a reason why not all antibodies would be recycled?

A

mechanism is subject to saturation, only so many receptors can bind antibodies
if you have a lot of antibodies, not all of them will be recycled (when there is not enough receptor then the unbound ones will be degraded)

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44
Q

what antibody is not recycled in the FcRn process?

A

IgG3 because it cannot bind to the FcRn
leading to a very short half life

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45
Q

why does IgG3 have a very short half life?

A

gG3 (long hinge) cannot bind to FcRn leading to a very short half life (only circulates for a few hours
meaning it is degraded in the lysosome

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46
Q

what part of the antibody is responsible for binding the FcRn?

A

a monoaminoacid at the CH3 of the tail

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47
Q

how can the mechanism that extends the half life of antibodies be use therapeutically for autoimmunity?

A

isolation of antibodies from blood, taken 10000 donors, create a product and give a very high dose of these non-specific antibodies to people with autoimmunity
Bcos you saturate this mechanism any antibody including autoantibodies that would normally be recycled they would be degraded – just be offering a lot of non specific IgG – a useful therapy that didn’t cause a lot of side effects

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48
Q

what did paul ehrlich hypothesise in 1901?

A

“magic bullets” to target and destroy bacterial and infectious diseases (syphilis). In 1900s, a german physician interested in science came up with idea that theres a system of recognition, he came up with the magic bullet model

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49
Q

who was responsible for the idea of monoclonal antibodies for immunotherapy?

A

Milstein and Kohler (1975)

50
Q

who won a nobel prize for cancer immunotherapy in 2018?

A

Jim Allison (2018)
(blocking CTLA4)

51
Q

the global market for monoclonal antibody therapeutics is predicted to bring how much money in 2023?

A

$218.97 billion

52
Q

how do you create a hybridoma?/ produce a monoclonal antibody
against an antigen

A
  1. mouse is immunised with antigen X and the mouse spleen produces plasma cells that secrete antibodies against the antigen
  2. tumour (myeloma) cells (that don’t produce antibodies) are selected
  3. plasma cells are taken from the spleen and lymph nodes
  4. culture these together with the tumour cells and selection marker and apply polyethylene glycol to allow fusion
  5. split cells into separate wells (one cell per well)
  6. if fused then the selection marker will allow them to survive (if not fused they die)
  7. these fused cells grow and multiple so we have a cell population that all produce specifci antibodies against antigen X (a monoclonal antibody) (with the same affinity and specificity)
53
Q

what is a monoclonal antibody?

A

A monoclonal antibody is a type of antibody produced from a single cell known as a hybridoma. All antibodies produced by the hybridoma are identical and bind to the same specific target in the same way.

54
Q

what was the outcome of the monoclonal antibody trail by Kohler and Milstein in 1975 ?
(OKT3)

A

OKT3 is an anti Cd3 monoclonal antibody created in mice and used to treat T cell keukaemia
the trial did not go well - there were alot of disadvantages
 No efficient interaction with the human immune system, recognized as foreign by the human body: leading to rapid clearance, especially upon re-treatment, allergic reactions
 Lots of anaphalatic reactions, wasn’t what anybody was hoping it would be – this occurred due to the fact it was a mouse antibody, so immune reactions against the it

55
Q

what way can we create fully human antibodies?

A

using phage display or germline sequences, or mice that express human immunoglobulins
genetic engineering

56
Q

what ways can we create antibodies?

chimeric, humanised, human

A

Genetic engineering
V gene cloning
CDR grafting
Eukaryotic expression

57
Q

who came up with the technology for chimeric antibodies?

A

Micheal Neuberger and Terrence Rabbits in 1984

58
Q

who came up with the technology for humanised antibodies?

A

Greg winter in 1986

59
Q

after you isolate the B cells form an immunised mouse, what two things can you do to engineer an antibody

A
  • generate a hybridoma to create monoclonal antibodies
  • directly isolate mRNA from B cells, create cDNA, amplify varibale heavy and light chains and then generate recombinant antibodies and subclone into a vector for expression in ecoli
60
Q

why do you need two plasmids to create a antibody?

A

one for the light chain and one for the heavy chain

61
Q

what things can alter when engineering antibodies from VH and VL DNA sequences?

A
  • switch species
  • switch classes (gG, gA or D)
  • switch subclasses
  • switch formats (just fab or scFv or trispecific etc)
62
Q

describe a method to produce a fully human antibody

A

Need access to human antibody library – germline library
PCR heavy and light chain
Transform into bacteria
Use phages to transfect into bacteria
The phages will take up the genes expressing the antibody (svfv)
All the phages expressing diff antibody fragments
Using a panning system you can see if any phages have the specificity of you choice
Coat antigen onto solid phase and flow over phages – if any interaction the phages will bind and the rest are washed off
This process is repeated a few times
Isolate the scfv and variable light and heavy component and subclone them in IgG expression vector and transfect into mammalian cell leading to secretion of fully function human antibody

63
Q

what display techniques can be used to create a fully human antibody?

A

Phage display
Yeast display
Ribosome display

64
Q

give three techniques to produce fully human antibodies

A

microbial surface display
transgenic mouse
human memory B cell immortalisation
humanisation

65
Q

how can transgenic mice be used to create fully human antibodies?
what are the benefits?

A

Human antibody-producing mouse strains are generated by introduction of human heavy- and κ-chain minigene constructs into the germline of lg-inactivated mice

by using transgenic mice that express human immunoglobulin you can create fully human antibodies -immunise them with antigen then generate hyrbridoma which will produce antibodies

avoid the subsequent costly and tedious in vitro affinity maturation and antibody humanization process.

66
Q

what are the benefits of using a transgenic mouse to produce human antibodies?

A
  • Rapid generation of fully human high affinity antibodies
  • Less potential allergic responses or other side effects (HAMA, HACA)
  • No humanization or complicated genetic engineering, which can be time consuming and expensive.
67
Q

what is humira?

A

Adalimumab
A human antibody produced by phage display targeting TNFalpha
used therapeutically for rheumatoid arthritis and other inflammatory disease

developed by Greg Winter

68
Q

what are single domain antibodies? what are the benefits

A

dAbs have only a single chain (single heavy and light chain) - still has specificty but is much easier to get through membranes and inside cells

69
Q

when was the first antibody chimerised?

A

1984

70
Q

when was hybridoma technology developed?

A

1975

71
Q

what was the first chimeric antibody?

A

rituximab

72
Q

what is Alemtuzumab?

A

Alemtuzumab is a monoclonal antibody that targets a protein called CD52 on the surface of most types of white blood cells
Alemtuzumab is a type of targeted cancer drug. It is a treatment for several cancers including: chronic lymphoblastic leukaemia (CLL), T-cell prolymphocytic leukaemia (T-PLL), skin lymphoma
Doctors also use alemtuzumab as part of a stem cell transplant to reduce the risk of a complication called graft versus host disease

73
Q

what is C1q?

A

complement component 1q (or simply C1q) is a protein complex involved in the complement system, which is part of the innate immune system.

When C1q binds antigen-antibody complexes, the C1 complex becomes activated. Activation of the C1 complex initiates the classical complement pathway of the complement system. The antibodies IgM and all IgG subclasses except IgG4 are able to initiate the complement system.

74
Q

what ways can antibodies target tumour cells

A
  • antibodies can bind to protein and activate the complement system to clear the target cell
  • antibodies can interact with fc receptors on immune cells like macophages to promote phagocytosis
  • antibodies can directly bind an antigen and cross link to cause apoptotic signalling (eg herceptin)
  • antibodies can neutralise or scavenge some ligands to neutralise receptors
  • antibodies can target cells/proteins in the tumour microenvironment (ie removing inhibitory signals) to enhance the immune system
75
Q

why is CD20 an ideal target for antibody therapy?

A

if an antibody binds to CD20 then distribution changes occur and the antibody pulls the cd20 into the rafts, forming perfect platform for complement to bind (bcos antibodies are clustered) but also induces apoptotic singal that leads to cell death
CD20 is close to the surface of the membrane and has an extracellular loop
when cd20 is knocked out in mice there are no adverse effects

76
Q

what are membrane rafts composed of?

A

cholesterol and shpingolipids make up the raft and various functionally related molecules sit on it

77
Q

what properties are unque to anti Cd20 antibodies (rituximab)?

A
78
Q

describe the experiment that proved the importance of Fc receptors in rituximab therapy/ in cell killing

A

Experimentation with nude mice and inject tumour cells into mouse, follow the size of the tumour
In wt mice treat with pbs, the tumour grows rapidly (control)
Animal treated with rituximab were protected from this
However in animal that don’t express fc receptor theres no longer protection by the antibody
You need fc receptors for the antibody to work – complement and fc recptors are required for function of rituximab

79
Q

what is the mechanism of action for rituximab?

A

binds to the larger loop of CD20 causing redistribution of the ocmplex to lipid rafts
this activates complement (due to clustering of antibodies/CD20 on membrane rafts) causing complement dependent cellular cytotoxicty and also causes apoptosis
macrophages can also bind to the antibody by their fcreceptor inducing phagocytosis
NK cells carry a different receptor that recognises the antibody causing degranulation causing killing

80
Q

what is event free survival?

A

In cancer, the length of time after primary treatment for a cancer ends that the patient remains free of certain complications or events that the treatment was intended to prevent or delay.

81
Q

when did research start and when was rituximab approved?

A

first research started in 1980s, approved by FDA in 1998

82
Q

what is Ofatumumab?

A

fully human anti-Cd20 mAb
Binds slightly different, to the smaller loop with higher affinity and higher complement activation

(being used as a treatment for multiple sclerosis relapse in adults but will be approve for CLL later this year)

83
Q

what ways can resistance develop from antibody therapies?

A
  • Monocytes/ Macrophages interact with the protein and shave off the whole complex from its membrane so its not internalised but removed (Trogocytosis)
  • Complement depleted – there is not enough complement around to activate the antibodies
  • Increased expression of regulatory protein that protect tumour cell from CDCC – tumour adapts to increase this
  • Changes in tumour environment
  • Reduced fc receptor expression
  • Changes in signalling
84
Q

what is trogocytosis?

A

a process in which one cell physically extracts and ingests “bites” of cellular material from another cell

85
Q

how does resistance to rituximab develop?

A

Change in expression of CD20
(when antibody binds whole complex can be internalised which cause reduced expression of target so less Ab can bind)

86
Q

what are the effects of TNF in rheumatoid arthritis?

A
87
Q

whats the difference between a type 1 and type 2 antibody inhibitor?

A

type 1 draws the molecule into a lupid raft for FcR2b activation and CD20 internalisation
type 2 binds but no lipid raft localisation, fcr2b activation or cd20 internalisation

88
Q

combination therapy with what target enhances rituximab clinical effects

A

CD27 stimulation enhances anti-tumor efficacy of tumor-targeting mAbs

89
Q

how does CD27 stimulation enhance antitumour efficacy?

A

Stimulation of CD27 on T and NK cells increases chemokine (CCL3, 4,5) and IFNg release as well causing t cell proliferation
Released IFNg leads to myeloid cell activation and infiltration
Activated macrophages have enhanced anti-CD20 dependent phagocytic capability

CD27, a costimulatory receptor expressed constitutively on T and NK cells

90
Q

what is cd27?

A

a costimulatory receptor expressed constitutively on T and NK cells

91
Q

whats the difference between osteoarthritis and rheumatoid arthritis?

A

Osteoarthritis occurs when the smooth cartilage joint surface wears out. Osteoarthritis usually begins in an isolated joint. Thinned carilage and bones ends rub together.

Rheumatoid arthritis is an autoimmune disease, which means that the immune system malfunctions and attacks the body instead of intruders. swollen inflamed synovial membrane and bone erosion

92
Q

what are the roles of TNF in immune regulation?

A

TNF is important in the maturation of dendritic cells.
TNF is important in the host defense against organisms that induce granuloma formation or that exist intracellularly in monocyte-macrophage cells
TNF a controls humoral autoimmunity.

93
Q

lack of TNF increases suseptibilty to what pathogens

A

mycobacteria, fungi, and Listeria.

94
Q

why is there increased incidence of TB following TNF blocking agents?

A

Lack of TNF increase suseptibilty to mycobacteria (TB = mycobacterium tubulercolis)
due to TNFs importance in the host defence against organisms that exist intracellularly in monocyte macrophage cells
alveolar macrophages (AMs) being the primary conduit of infection and disease

95
Q

why are TNF blcoking agents not recommended for MS?

A

There are several theories about how anti-TNF therapy might risk MS in certain patients. Aviña-Zubieta speculated that the therapy may increase reactivity from immune cells to myelin leading to a loss and malfunction of the affected areas

96
Q

what are the five tnf inhibitors that have been approved by the FDA?

A

etanercept, infliximab, adalimumab, golimumab. and certolizumab

97
Q

what are the effects of TNF in rheumatoid arthritis?

A

monocyte secretion of TNF activates three cell types, endothelial cells, synovial fibroblasts and monocytes
it causes endothelial cells to upregulate surface adhesion molecules increase infiltration of immune cells(t cells monocytes)
it causes monocytes to secrete ODF that activates osteoclasts. they also secrete IL1 which activates both T cells and synovial fibroblasts
synovial fibroblasts secrete IL8 that helps stimulate migration through endothelium into synovial tissues. In addition they secrete matrix metalloproteinases, prostaglandin E and IL6 which attack cartilage

98
Q

tnf alpha inhibitors are approved for treatment of what diseases?

A

TNF-alpha inhibitors are authorised for the treatment of inflammatory and autoimmune conditions, such as rheumatoid arthritis, ankylosing spondylitits, Crohn’s disease, ulcerative colitis, psoriasis, and psoriatic arthritis

99
Q

what happens to the synovial membrane in rheumatoid arthritis?

A

As rheumatoid arthritis progresses, the synovium, which produces synovial fluid, swells and thickens, producing an excess of synovial fluid. This, in turn, leads to further swelling and inflammation which causes pain and stiffness in the joint.
The inflamed synovium can eventually invade and destroy the cartilage and bone within the joint.

100
Q

what is rheumatoid arthritis?

A

an autoimmune and inflammatory disease, which means that your immune system attacks healthy cells in your body by mistake, causing inflammation (painful swelling) in the affected parts of the body. RA mainly attacks the joints, usually many joints at once.

101
Q

what are the potential functional properties of TNF inhibitors?

A

LTalpha binding
soluble TNF binding
soluble TNF neutralisation
membrane TNF binding
membrane TNF agonist
FcR binding
Fc mediated ADCC/CDC
inhibits cytokine synthesis

102
Q

other than TNF inhibitors what treatments are available for RA?

A

Nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen. Corticosteroids, such as prednisone. Conventional DMARDs (Disease-modifying antirheumatic drugs), which can slow RA progression and possibly save the joints from permanent damage

103
Q

how can we make antibodies better (in terms of therapy)?

A
  • subtype switching (IgG1, IgG2, IgG3, IgG4)
  • species switching (human, mosue, rat, rabbit)
  • isotype switching (IgG, IgE, IgM, IgA)
  • reformatting (diabody, Fab, bispecific, engineeredFc)
104
Q

what two amino acids in CD20 are important for antibody binding?

A

Alanine and proline at the top extracellular loop of CD20 are important, highly targeted by antibodies because of extracellular binding close to the surface of the membrane

105
Q

what are the two application s for the pFUSE plasmids (heavy chain and light chain plasmids)

A

mAb isotype switching for varying effector functions
whole recombinant antibody production with cotransfections

106
Q

what is receptor mediated transcytosis?

A

receptor-mediated transcytosis (RMT) is a vesicular transcellular route by which various macromolecules are transported across a barrier
can occur accross the blood brain barrier - can aid therapeutic antibodies transport through the BBB

107
Q

What is the potential impact of mutating the V domain sequences using display libraries and/or rationale design

A

altered binding affinity or specificity

108
Q

what are the potenial impacts of mutating Fc sequence using display libraries and/or rationale design: select IgG isotype

A

Increase/decrease ADCC
Increase/decrease ADCP
Increase/decrease CDC

ADCC - antibody dependent cellular cytotoxicity
ADCP - antibody dependent cellular phagocytosis
CDC - complement dependent cytotoxicity

109
Q

what are the potential impacts of mutating Fc sequence usign design libraries and/or rationale design?

A

increase or decrease half life

110
Q

what are the potential impacts of an antibody fragment lacking Fc?

A

decrease half life, decrease CDC, decrease ADCC, decrease ADCP

111
Q

give three glycosylation strategies for modifying FcgammaR and complement interactions

A

A glycosylation
Bisecting N-acetylglucosamine
Non fucosylation

112
Q

what are the potential impacts of A glycosylation of antibodies

A

decreases ADCC, decrease ADCP, decrease CDC

113
Q

what are the potential impacts of Bisecting N-acetylglucosamine or Non-fucosylation on antibodies?

A

increase ADCC

114
Q

what amino acids are critical to allow IgG4 antibodies to engage in Fab arm exchange?

A

serine at position 228 in the hinge
and
arginine at position 409 in the CH3 domain

114
Q

what amino acids are critical to allow IgG4 antibodies to engage in Fab arm exchange?

A

serine at position 228 in the hinge
and
arginine at position 409 in the CH3 domain

115
Q

What technology was used to create the first generation bispecific antibody?
What year?

A

Quadroma
~1980

116
Q

what was the problem with quadroma technology?

A

Bispecific antibodies were generated by combining the light and heavy chains of two sifferent monoclonal antibodies. the two antigen bidning sites of the rresulting antibody are aiming at different targets of interest.
It produces a mixture of ten different antibodies. this pakes purification of the one wanted antibody out of the micture very complex.

117
Q

what problem did ‘knob into hole’ technology solve and when?

A

solves the porblem of heavy chain mispairing
in 1997
with specific pairing of the heavy chains only four possible products are formed reducing the purification complexity

118
Q

how did CrossMAb technology revolutionise bispecifc antibody production?

A

it solves the problem of light chain mispairing
By exhcnaing the ‘molecular bricks’ between heavy and light chains only specific interactions may take place at his region too selectively producing the desired bispecific antibody
only the desired antibody is produced

119
Q

what is the industry average success rate of drug development from pre clincial to launch?

A

4.1%

120
Q

what is the overall success rate of alzheimers disease drug development from preclinical to launch?

A

0.5%

compared to the 4.1% industry average

Biomedical technology (9 decks)

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