Large Molecule Drugs

Healthy mouse exposed to a deadly virus (e.g., bird flu) may die due to virus replication.
If the mouse is injected with an inactivated form of the virus (e.g., heat-inactivated), it survives.
Later injection with the active virus leads to survival, demonstrating acquired immunity.
Taking serum from the immune mouse and injecting it into a naive mouse provides passive protection due to transferred antibodies.

Vaccine: Trains the immune system using an inactive or attenuated virus to prepare for future infections.
- Can also involve delivering RNA or DNA to induce antibody production.
Therapeutic: Provides pre-made antibodies to protect against a specific threat; passive immunization.

Using antibodies as medicine is not a new concept (dating back 150 years).
Early methods involved growing antibodies in animals (e.g., horses) and isolating them for therapeutic use.
Example: Rattlesnake antivenom produced by injecting venom into horses, then isolating the antibodies from the horse blood.

Uses antibodies from recovered patients to treat others. Example: Using serum from Ebola survivors during the 1970s and 2014 outbreaks.

Soluble proteins with a characteristic Y shape.
Composed of two heavy and two light chains.
Molecular weight is approximately 150 kDa.
Produced and secreted by B cells.
Two Key Components:
- Variable Region (Fab): The arms of the antibody, with highly diverse sequences determining binding specificity.
- Constant Region (Fc): The bottom portion, interacts with other immune system components.

Specificity arises from Complementarity Determining Regions (CDRs), loops on the Fab regions.
- Three CDRs on the heavy chain and three on the light chain.
Antibodies can target surface proteins on bacteria, viruses, or cancer cells.
Can also target healthy proteins, leading to autoimmune diseases.

IgG: Most abundant in serum (~90%), long half-life (21 days), crosses the placenta.
IgM: Pentamers or hexamers (multiple IgG-like components).
IgA: Mucosal immunity (gut lining, nose).
IgE: Targets parasites and implicated in allergies; binds allergens like dust mites.

Focus of biotherapeutics due to abundance, long half-life, and ease of production.

Neutralization: Blocking pathogens or toxins by binding to them.
- Example: Antibodies binding to cholera toxin in the intestine.
Agglutination/Aggregation: Clumping pathogens together via multiple antibody arms.
- Example: Antibodies clumping bacteria cell surface proteins.
Fc-Mediated Effector Functions: Engaging other immune cells via the Fc region.
- Recruiting macrophages to phagocytose bacteria.
- Activating complement to attack antibody-labeled cells.
- Recruiting natural killer cells to kill antibody-labeled cells.

B cells are white blood cells originating in bone marrow.
Each B cell has a B cell receptor (BCR), an antibody-like molecule on its membrane.
B cells that react to self-proteins are eliminated to prevent autoimmunity.
When a BCR binds a foreign antigen (e.g., influenza virus), the B cell activates, divides rapidly, and mutates to improve binding.
Activated B cells differentiate into:
- Plasma cells: Antibody factories producing soluble versions of the BCR.
- Memory cells: Dormant cells that provide rapid response upon subsequent exposure.

Injecting an animal (e.g., horse, mouse) with an antigen stimulates antibody production.
Blood is then collected, and antibodies are isolated.
The resulting antibody mixture is polyclonal, meaning it contains a variety of antibodies from multiple B cell clones.
Polyclonal antibodies are a mixture, with varied specificities and affinities, but can still be useful.

Example of Gamestan, antibodies from donors vaccinated with MMR that have high antibody titers for measles.

Mouse injected with antigen, B cells harvests from spleen, and hybridized with tumor cells to create a hybridoma to make them immortalized.
Monoclonal antibodies are derived from a single B cell clone.
Because they work by a single sequence, they are produced from one clone.
Recognize a single protein at a single surface.
Example: Nirsimumab, for example, is an anti-RSV antibody administered to infants. A newer version extends the half-life to 100 days.

Recombinant technology is used, involving the following steps:
- Light and heavy chain DNA sequence known and put into a vector.
- Transfect that into cells.
- Mammalian cells (e.g., Chinese hamster ovary (CHO), HEK 293) are used for authentic antibody production.
- Cells are grown up on liters or galons in bioreactors.
- Quality Control is completed to make sure the batch is not infected, in which case the company would lose money.
  - $50,000,000$
There's interest in using plants like tobacco to produce antibodies cheaply (viral vectors deliver genes to crops).

Purification removes host cell proteins like Protein A or Protein G, minimizing unwanted material.
Large and heterogeneous, modifications, not always chemically pure.
Quality control involves acceptable levels of modifications such as N-terminal modifications, glycosylation.
Unlike small molecules, antibodies have limited shelf life and may require deep-freeze conditions.

Approved in 1986 to prevent transplant rejection.
Antibodies are the fastest-growing class of therapeutics, including biosimilars (generics) and biobetters (improved versions).

In the 1970s, it was created to create monoclonal antibodies.
Murine monoclonal antibody was made from the clone OKT3 and had antiCD3 material.
T cells are responsible for tissue rejection. The idea was to shut down T cell activity to prevent organ rejection.
During a bolus of the antibody right before the transplant, and for two weeks after, T cells are prevented from coming back.

Binds to the CD3 receptor on T cells, crosslinking T cell receptors via bivalent binding.
Simulates high levels of foreign antigen, causing T cells to kill themselves, knocking them out of cell circulation.

Proven to work on kidney transplants.
The antibody is from a mouse, which can cause a pre-existing allergy and trigger an adverse reaction.
Injection leads to natural antibodies against it clearing out the therapeutic antibody.
Can only be used once because of being inoculated against it.

Specific, targets easily designed, may or may not be effective.
Hard to predict because of parts of the immune system still not entirely understood.
Expensive and harder to develop and manufacture than other classes of drugs, promising and fast growing out there.