Lecture 20: Antibody Drugs Part 1- Intro

Around what time of history do antibody biologic drugs begin to impact pharmacy?

Early 2000s

Describe Antibodies

‣ Proteins functioning as key part of immune system

‣ Large proteins designed to bind with extremely high affinity and specificity to a specific target -> antigen

‣ Antibodies (aka immunoglobulins (Ig)) are a class of glycoproteins (protein+carbohydrate) made by immune cells to target specific proteins or other molecules

‣ Antibodies weaponize specific proteins/protein interactions to protect host organism

Describe antibody drugs

Antibodies targeted to an intentional therapeutic target

What are newer antibody biologic drugs composed of?

They are single molecule

How are newer antibody biologic drugs mass produced?

Mass produced using recombinant protein expression and purification methods

-> mAb

What is the main takeaway about antibody biologics that the lecture emphasizes?

Antibody biologics are potential "magic bullets" ruthlessly binding one target, including many undruggable targets with small molecule drugs

What are the 2 disease states where antibody drugs are the primary therapeutics?

Cancer and autoimmune/inflammatory conditions

What is the main trigger for auto-immune diseases?

Auto-immune diseases are largely caused by abnormal immune system signaling

How do antibody drugs work to help treat auto-immune disease?

Antibody drugs can shut off that pathological immune signaling either by:

- binding the soluble immune protein (ie, cytokine)

- binding the cell surface receptor

What is the main cause of cancer as explained in the lecture?

A disease of immune system failure

- immune system designed to recognize and destroy abnormal cells

- in cancer, the immune system is "blind" to the mutated cells

How do antibody drugs help to treat cancer?

Antibody drugs can bind to cancer cells

- engage the immune cells to destroy the cancer

What are antibodies part of?

Immune system

Where does the immune system primarily work?

Outside of cells

How does the immune system work?

‣ Immune system largely works through signaling proteins (cytokines, antibodies) interacting with proteins on the cell surface

‣ Immune system makes many soluble proteins and signaling molecules that work through binding to the outside of the cells, at cell surface receptors

What is another function of cell surface receptors other than binding signaling molecules?

Also used by immune cells to recognize and bind to certain cells

Where do antibodies bind to?

Antigens

Define Antigen

Specific target that an antibody binds to with extremely high affinity and specificity

What is the chief function of antigens?

Bind foreign or unnatural/mutated proteins

- esp. important in host defense against infectious organisms

What is the source of antibodies (2)?

1. Loaded on surface of the immune cells

2. Secreted by immune cells as soluble proteins to patrol the blood and other fluids

What happens when an antibody binds to an antigen?

Covers and inactivates the antigen

- signals immune system to develop response to targeted antigen

What are the 5 different types of antibodies?

IgM, IgA, IgG, IgD, IgE

- IgG is the focus for biotechnology

What is the typical antibody structure?

3 parts:

- heavy chain protein

- light chain protein

- carbohydrate (glycan) chain

Describe heavy chain proteins in antibodies

Dimer of heavy chain proteins

-crosslinked together via disulfide bonds

How do the light chains and heavy chains interact?

Each of the heavy chain proteins interacts with a separate, smaller protein -> light chain proteins

- glycosylated at one position with chain of carbohydrates -> glycan chain

What bonds are responsible for binding heavy chains to each other and to light chains?

Disulfide bonds

Define glycoprotein

A protein with 1+ carbohydrate chains attached to amino acid side chains

What is glycolsylation?

Glycosylation of protein = adding glycan chain

- example of PTM

What glycosylation occurs in the IgG antibody?

‣ Asparagine 297 (Asn297) is glycosylated with glycan chains

- Asn297 is in Fc domain

‣ Glycan change made of multiple different types of carbohydrates

- chains + branched chaina

Why is the glycosylation in the IgG antibody crucial?

The change is essential for the Fc domain to bind to cell surface receptors that recognize the Fc domain

- also stabilized the antibody protein structure

- increasing half lives

What do antibody drugs typically have that resemble the IgG antibody?

Glycan chains

What are the two functional antibody fragments?

1. Fragment antigen-binding (Fab)

2. Fragment Crystallization (Fc)

How were these fragments discovered?

Early in the history of studying antibodies, protease named papain was discovered to be able to cut antibody protein

- Papain cuts the antibody in a flexible region of the heavy chain

-> hinge region

What was the result of cutting the hinge region?

Production of 3 different protein fragments

- 2 identical pieces, 1 individual

Summary: 4 total proteins (2 HC, 2 LC) cut to produce 3 protein fragments

What fragment makes up the 2 identical twins?

Fab; Pair of identical twins were found to be able to still recognize the antigen

Describe Fab

‣ Two upper arms that are responsible for recognizing antigen

‣ Retains antigen recognition after being cleaved by papain

What made up the remaining individual fragment?

Fc portion; The remaining fragment was discovered to be highly crystalline, crystallized readily

Describe Fc Portion

‣ Lower stem that's highly stable, capable of forming protein crystals

‣ One fragment produced if cut by papain

‣ Fc domain also binds to cell surface receptors

What is Fc used for now a days and why?

Stabilizing GLP-1 peptide; Fc domain is such a stable protein, it is used to make stable protein

What are the 4 domains of Heavy chain?

‣ 3 constant domains

‣ 1 variable domain (Vh)

How many domains does a light chain have?

2: one constant and one variable (VL)

Which domains are identical in all antibodies of a given class?

Constant domains

What is the purpose of the variable regions?

Built by the B cells to recognize a single antigen

How may antigens can each antibody recognize?

One antigen

- each antibody has 2 variable regions

Describe Epitope

The specific region of an antigen that is recognized by the variable domains of the antibody

How many epitopes can a single antigen have?

Several

Are those epitopes recognized by the same antibody?

No

How many epitopes can an antibody bind?

Each antibody can only bind a single epitope

How does a variable region interact with an epitope?

Each variable region are both contacting and binding the epitope