1. Antibody Engineering

Question 1

Immunoglobulins:
Found in?
Secreted by?
Classes?
Recognise?
Trigger?

Answer 1

Present in plasma, tissues secretions, and lymphatics
Secreted by activated B cells (plasma cells)
5 classes (IgA, IgD, IgE, IgG, IgM)
Recognise antigen
Trigger effector function

Question 2

Hypervariable loops/Complentarity Determining Regions (CDRs)

Answer 2

Are found in both the light and heavy variable domains, the 6 CDRs form close-lying loop creating the structure of the antigen binding site

Question 3

2 arms/branches of effector function

Answer 3

1. Complement

2. Fc Receptors

Question 4

Subclasses of IgG:
Number
Names
What is the difference between subclasses
Where do the subclasses differ most

Answer 4

4
IgG1, IgG2, IgG3, IgG4
Each has a different gene encoding their heavy chain constant region
Their heavy chains differ in sequence, most notably in the hinge region

Question 5

Subclasses of IgG:
Number
Names
What is the difference between subclasses
Where do the subclasses differ most

Answer 5

4
IgG1, IgG2, IgG3, IgG4
Each has a different gene encoding their heavy chain constant region
Their heavy chains differ in sequence, most notably in the hinge region

Question 6

Classical pathway of complement activation

Answer 6

IgG interact with antigenic surface

Then IgG molecules interact via their Fc regions, creating a platform for C1q to bind to, enabling further complement activation

Question 7

Human IgG subclasses vary in their ability to bind C1q and activate complement

Answer 7

IgG1: +++
IgG2: +/-
IgG3: +++
IgG4: -

Question 8

Human FcγR
Specific for which Ig?
Signalling motifs in the γ chain?
Where on the Ig does FcγR interact?

Answer 8

FcγR is specific for IgG

γ chain possesses either ITAM (Immunoreceptor Tyrosine-based activation motif) or ITIM (Immunoreceptor Tyrosine-based inhibitory motif) signalling motifs

Interacts with the lower hinge region on IgG

Question 9

Fc Receptor Function

Answer 9

1. Phagocytosis (more efficient when target is opsonised with IgG)
2. Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)
3. Respiratory burst (release of activated oxygen species)
4. Release of inflammatory mediators and enzymes

Question 10

Monoclonal Antibodies
Description?
Characteristics?
Created how?

Answer 10

A homogenous preparation of antibody

All molecules are identical: They are of a single Ig class and have a defined affinity for an antigen target

Traditionally produced by hybridoma technology

• Spleen cell from mouse + immortal myeloma cell fused with PEG -> Hybridomas
• Unfused cells die, Hybridomas survive -> mAbs generated as surviving/selected cells secretes mAbs

Question 11

Problems with using mouse monoclonal antibodies to treat humans:

Answer 11

1. Immunogenic in humans -> HAMA response (Repeated doses may result in serum sickness)
2. Relatively quick clearance of mouse mAbs in humans
3. Unconjugated mouse mAbs are poor at recruiting human effector systems (FcγR and complement)

Question 12

There exist problems with using mouse monoclonal antibodies to treat humans, so how did scientists get around this issue?

Answer 12

Problems with mAbs so developed
-> Chimeric Human/Mouse antibodies

However still immunogenicity problems so developed
-> Humanised antibodies (via CDR grafting)

Still not perfect however, so scientists look towards creating actual “Human antibodies”

Question 13

Modulating IgG effector function through Fc engineering

[4 ways effector function may be changed]

Answer 13

1. Increasing effector functions through Fc engineering
2. Decreasing effector functions through Fc engineering
3. Enhancing serum half-life of IgG through Fc engineering
4. Glycoengineeering to influence effector function

Question 14

Modulating IgG effector function through Fc engineering

[4 ways effector function may be changed]

Answer 14

1. Increasing effector functions through Fc engineering
   - Focus on increasing the affinity of the Fc of IgG for the low affintiy receptors (such as FcγRIIIa)
2. Decreasing effector functions through Fc engineering
3. Enhancing serum half-life of IgG through Fc engineering
   - IgG naturally persists for a prolonged period in the serum due to FcRn-mediated recycling, typical half-life of 21 days
   - Efforts to engineer pH-dependent interaction of IgG Fc with FcRn receptor to increase affinity at pH 6.0 while retaining minimal binding at pH 7.4
4. Glycoengineeering to influence effector function
   - Loss/removal of oligosaccharides -> greatly reduced affintiy for FcγR
   - Afucosylated (non-fucosylated) IgG exhibits greatly enhanced ADCC activity through increased binding to FcγRIIIa