Immunoglobulin/Antibody (F)

Describe the structure of Immunoglobulin

Describe the structure of Immunoglobulin

4 Polypeptide Chains • 2 Light (L) Chains • 2 Heavy (H) Chains

Held Together By Disulphide Linkages

Heavy chains are larger and contain more genetic information.

Describe the variable domains of Immunoglobulin

Found on the N terminal (ends of the Y), Variable regions promote Ag Binding 3 hypervariable regions form loops that bind Ag based on complementarity - Complementarity Determining Region (CDR) The remaining V domain regions are structurally conserved Framework Regions (FW)

Describe the constant domains of Immunoglobulin

Found on C terminal, constant region promotes Effector Functions

Define: Idiotype

A collection of antigenic determinants contained in the antigen-binding V region. Each individual determinant is known as an idiotype. Immunoglobulin against different antigens have differing idiotypes.

How is antibody activity regulated?

Anti-idiotype antibodies (anti-IgM, anti-IgG etc) bind to the idiotypes of our own antibodies to prevent antigen binding (useful to end Ab activity when an infection is going away)

Define: Allotype

Subtle amino acid differences within the same constant region between individuals of the same species. For example IgG1, IgG2, IgG3, etc.

Define: Isotype

5 Classes of heavy chain constant region antigenic determinants: IgA, IgG, IgD, IgM, IgE. 2 Classes of light chain constant region antigenic determinants k, l.

Explain: IgG

• The most abundant immunoglobulin in sera, lymph, cerebrospinal fluid, and peritoneal fluid. 80% of serum immunoglobulin.

• Roles: activates complement, functions as an opsonin by binding to Fc receptors

• 4 subclasses IgG1, IgG2, IgG3, IgG4.

Define: Opsonin

Extracellular proteins that, when bound to substances or cells, induce phagocytosis.

Rank the IgG subclasses in their ability to activate complement

IgG3>IgG1>IgG2>IgG4

IgG3 is the most flexible, thus the best at activating complement.

Explain: IgM

• The first immunoglobulin produced

• Makes up 5-10% of serum immunoglobulin (lots is secreted)

• Often found in a pentameric soluble form

• Polyvalent binding agglutinates antigen and activates complement (high avidity)

• J-chain allows for secretion

Explain: IgD

• Makes up 0.2% of serum

• Found on the surface of mature B-cells with IgM (may function in the activation of B-cells)

• Function is unknown

• Single hinge is prone to cleavage (breaks down in blood)

Explain: IgA

• Minor serum immunoglobulin (5-10%)

• The most produced immunoglobulin (75% of total antibody production)

• Most predominant immunoglobulin in secretions

• Serum IgA is monomeric

• Secreted IgA is dimeric (+ J chain) \

• Two subclasses IgA1, IgA2

Explain how IgA1 and IgA2 differ

IgA1 is found in serum, 30 amino acids longer (larger hinge), and often cleaved when secreted. IgA2 is found in secretions, has a shorter hinge, and is thus protected in secretions containing bacteria.

What is required for antibodies to be secreted?

Binding by the J-chain which assists in transcytosis through the epithelial layer into the lumen.

Explain: IgE

• Smallest contribution to serum immunoglobulin

• No hinge region

• Binds to Fc receptors on basophils and mast cells to promote allergic reactions (mediate immediate hypersensitivity reactions resulting in hay fever, asthma, hives, anaphylaxis)

How can IgE reactions be subdued?

Desensitization Prolonged exposure to small doses of allergen

1. Could silence cells that produce the recognized allergen

2. Body may stop making IgE and make IgG instead.

Immunotherapy

• Tags all IgE with anti-IgE antibody and promotes phagocytosis (removes all IgE)

What do immunoglobulins do?

1. Act as B-cell receptors

2. Activate Complement

3. Neutralize

4. Fc Receptor Binding

Explain the role of immunoglobulin as B-cell receptors

Surface immunoglobulin (sIg) has short cytoplasmic domains and cannot mediate intracellular signals. sIG associates with the Ig superfamily members Iga and Igb which generate intracellular signals through Immunoreceptor Tyrosine-based Activation Motifs (ITAM).

If surface immunoglobulin bind antigen, but Iga and Igb are not present will the B-cell be activated?

No. To activate the B-cell sIg bind must Ag AND Iga Igb need to mediate the intracellular signal

How to Iga and Igb generate intracellular signals?

Immunoreceptor Tyrosine-based Activation Motifs (ITAM).

Explain the role of immunoglobulin in activating complement

Immunoglobulin bound to antigen can activate the classical pathway of the complement system if the complement component C1q binds to at least 2 Fc regions or 2 antibodies. (1 IgM or 2 IgG - within 40 nm of each other)

Explain the role of immunoglobulin in neutralization

High affinity Immunoglobulin (IgG, IgA) bound bacteria, viral particles, or toxins prevent their binding to target. Coating bacteria or virus with antibody will inhibit their ability to infect host tissues. Most toxins must bind a cell receptor to mediate detrimental effects → coating toxin with immunoglobulin protects host cells

Explain the role of immunoglobulin in Fc receptors

Several cell surface receptors can bind Ig Fc, Ig binds via its CH2 domain. Ig is bound by the a chain of the FcR. Signaling is mediated through a second molecule, the g chain (Except for FcgRII)

FcR binding can lead to cellular inhibition, activation, phagocytosis, induction of target killing and granule release.

In most cases, the Ig must be bound to antigen to stably associate with the FcR (except for IgE)

What are the effects of FcR binding?

Activation of respiratory burst Inhibition of stimulation Secretion/release of granules Degranulation Induction of killing Uptake Ab-dependent cell-mediated cytoxicity

Explain: Ab-dependent cell-mediated cytoxicity

A mechanism of cell-mediated immune defense whereby an effector cell of the immune system actively lyses a target cell, whose membrane-surface antigens have been bound by specific antibodies.

1. Coating of target with soluble Ig

2. Binding of NK cell to target via FcgRIII

3. Crosslinking of FcR induces targeted granule release (perforin + granzyme)

4. Death of target cell

Explain: Granulocyte Degranulation

Mast cells, basophils, and activated eosinophils express a high affinity IgE receptor (FceRI). Soluble IgE stably associates with the FcR in the absence of Ag binding.

Thus these cells are present, pre-coated in polyclonal Ab, ready to engage a variety of Ag.

Crosslinking of the FcR bound IgE by Ag induces degranulation.Granules release histamine and other pro-inflammatory mediators.

If the immunoglobulin on B-cells do not bind to antigen what happens?

Conventional B-cells are continuously produced throughout life. Most IgM+ IgD+ B cells (mature B-cells) that are newly produced have short half-life of 3-4 days. They must encounter antigen to survive, if not they die

What are some hurdles in antibody therapies? How can they be overcome?

Problems:

- The therapeutic Ab is too antigenic (humans see mouse antibodies as foreign and mount an immune response against the therapeutic antibody)

- Too short or too long of a half life

- Poor penetration into tissues

- Undesired or inefficient effector functions

By mutating / engineering the antibody genes in the hybridoma cell one can often reduce/eliminate this issues

What are chimeric Ab?

“Cloned” VJ and VDJ elements from a mouse Ab fused to the constant domains of a human Ab gene

What are humanized Ab?

The specificity of a given Ab is entirely determined by the 6 complementarity determining regions (CDRs) alone, as such the entire variable domains of mouse Ab are not required, just the CDRs.

What are some benefits of antibody therapies?

• Highly specific – less side-effects, collateral damage

• Customizable – can relatively easily retarget the therapy to a different cancer, pathogen or to keep up with evolving “bugs”

• Efficient – less drug needed

• Innovative – bispecific, soluble Fab fragments, antibody-drug conjugates

• Harness the immune system – may replace antibiotics to resolve the problem of multi-drug resistant pathogens