Therapeutic Applications of Monoclonal Antibodies

Cancer is a major application, but monoclonal antibodies are also used in other areas.
Considering the role of polyclonal antibodies and the reasons for treatment failure or relapse in some patients.
Exploring the potential of using monoclonal antibodies from recovered individuals to design vaccines.

Therapeutic Uses of Monoclonal Antibodies

Hematological Malignancies
Tumors
Asthma and Allergy
Autoimmune Diseases: Including rheumatoid arthritis.
Hypercholesterolemia and Cardiovascular Disease
Osteoporosis
Inflammatory Bowel Disease (IBD)
Graft Rejection
Infectious Diseases: Such as Ebola, COVID, malaria, and RSV.
Multiple Sclerosis
Paroxysmal Nocturnal Hemoglobinuria
Drug Reversal
The list of clinically approved applications is continuously growing.
Monoclonal antibodies are being trialed for numerous other conditions, aiming for more efficacious treatments.
The mechanisms of action include:
- Neutralizing to block infectious diseases.
- Killing cells associated with the disease process, like anti-CD20 for lymphoma and CLL.

Successes and Failures of Monoclonal Antibody Therapies

Many monoclonal antibodies fail due to not showing significant improvement over existing therapies.

Adverse Reactions

Adverse reactions are specific to individual antibodies rather than being a general property.
Antibodies are generally well-tolerated because they are a natural part of our circulatory system.
Some issues reported include:
- Unpredictable Effects: Such as the anti-CD28 disaster where an immunosuppressive antibody turned out to be immunostimulatory, causing severe immune activation and requiring amputations.
- Cardiotoxicity
- Infection Sensitivity: Due to modulation of the immune response.
- Acute Anaphylaxis: In response to repeated doses in some individuals.

Checkpoint Inhibitors

Checkpoint inhibitors balance attacking cancer and preventing the immune system from attacking itself.
Most patients on checkpoint inhibitors show signs of autoimmunity, often affecting the skin and intestines.

Reasons for Treatment Failure

Anti-Monoclonal Antibodies: Leading to loss of efficiency or anaphylaxis.
- Directed against chimeric parts or the binding site (anti-idiotype).
Target Loss: Cancer cells can lose the target molecule, rendering the monoclonal antibody useless.
Immune System Exhaustion: The recipient's immune system becomes exhausted and unresponsive to checkpoint inhibitors.
Fc Receptor Polymorphisms: Prevent effective activation of monoclonal antibody functions.
Tumor Microenvironment: Can make tumors resistant to immune penetration and activation.

Minimizing Problems and Antibody Properties

Modifications to antibodies and protocols aim to minimize these issues.
Antibodies are well-tolerated and stable with a long half-life (e.g., IgG about 20 days) compared to small synthetic drugs (hours).

Cost of Monoclonal Antibody Therapies

Cost is a substantial issue. Drug companies sell them at a premium to recover development costs.
High-use monoclonal antibodies can be very profitable.
Examples:
- Australia spent approximately $600 million on immune checkpoint inhibitors in 2019.
- Anti-TNF antibodies for rheumatoid arthritis treatment cost up to $320 million.
The global therapeutic monoclonal antibody market was worth $334 billion in 2021.

Monoclonal Antibodies as Therapeutics

Properties that make them adaptable to therapeutic design and development.
The potential advantages of bispecific antibodies over separate antibodies.
Understanding the "magic bullet" concept and its relation to other drugs.

Mechanisms of Action of Therapeutic Antibodies (Focused on Cancer)

Antibodies Used Directly: Target molecules on cancer cells to activate the immune system, leading to cell death via antibody-dependent cell cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).
Changing Cell Behavior: Binding to surface molecules can induce cell death, such as CD3 on T cells.
Antibodies as Drug Delivery Vehicles: Attaching cytokines, drugs, radionucleotides, or liposomes to the Fc region for targeted delivery.
Bispecific Antibodies: Genetically engineered antibodies with one arm targeting cancer cells and the other targeting T cells for activation.
CAR T Cells (Chimeric Antigen Receptor T Cells): Single-chain fragment variable (scFv) from an antibody is grafted onto protein domains that stimulate T cell signaling, creating dedicated cancer-specific killer T cells.

Immune Response and Checkpoint Inhibitors

Immune responses to target cells are subject to inhibition to prevent them from getting out of control.
Checkpoint inhibitors target these inhibitory molecules to enhance immune responses to cancer.
Examples: PD-1 (discovered by Tsushima Honjo) and ipilimumab.

CAR T Cell Example

Remarkable remission in a patient with B cell tumor treated with CAR T cells targeting CD19.
The treatment effectively eliminated all B cells in the bone marrow and blood, demonstrating its efficiency.

Creating Mice with Human Immunoglobulin Genes

Human Ig genes are used to replace the variable region genes of the mouse immunoglobulin heavy and light chain loci.
When immunized, the mouse makes a human-like immune response.
Hybridomas are created, and clones with the desired specificity are identified.
VH and VL genes are cloned and reconstituted with fully human constant region genes.
The Xenomouse (1996) is an example that sees human antigens as foreign, producing human antibodies.
Examples of antibodies developed: penitumumab (colorectal cancer) and ipilimumab (melanoma).
Velocimouse: human Ig genes have been targeted to the mouse loci. Chimeric antibodies produced.

Recovering B Cells from Immunologically Unique Humans

Idea to recover HIV-specific B cells from people who are naturally resistant to HIV to find antibodies important for that property.
Variants of the virus are used to "fish out" memory B cells that recognize those forms of the virus.
Identified cells are separated, grown, and assessed for the antibody they produce that is specific for HIV.
VH and VL genes are recovered, put into expression vectors, and a fully reconstituted antibody is made.
Used to create broadly neutralizing antibodies to HIV.
Limitations: cannot deliberately immunize humans with anything and they will not make immune responses to human proteins.

Avoiding Human Use: Phage Screens

Transfer the entire immune system into the laboratory.
B cells have been isolated and pooled together and all of the VH and VL genes from all of those B cells, from all of those people have been recovered by standard gene manipulations transferred into bacteriophage.
VH and VL joined together at random mimicking B cell development.
Phage expresses on its surface a VHVL combination allowing for the screening of libraries for binding to targets.
Process can be used to screen for higher and higher affinity binders.
Examples are adalimumab or Humira which is a huge importance in rheumatoid arthritis and belimumab which is an antibody for human B cell activating factor or BAF which is the survival factor for human B cells and this antibody is used in the treatment of lupus erythematosus.

Monoclonal Antibodies in Therapy: Considerations

Using monoclonal antibodies as therapeutics, considering their properties and adaptability.
Chimeric antibodies and the host immune response.
Tolerance in monoclonal antibody production.
Species used in making B cell donors and antibodies.
Diversity of systems available for making antibodies and their limitations.

First Monoclonal Antibody Used in Therapy (1986)

Specific for human CD3.
Mouse monoclonal antibody.
Aimed at depleting T cells in kidney recipients to prevent graft rejection.
Specific and high affinity.
Unlimited quantities.
Problems:
- Seen as foreign in humans and eventually rejected.
- Short serum half-life.
- Absence of key effector functions due to mouse-derived constant regions.
- Ineffective at depleting T cells.
T cells were killed by activation-induced death, leading to a cytokine release storm.
Humans recognized mouse and rat immunoglobulin as foreign

Making Antibodies More Human

Swap out domains by exchanging the mouse or rat parts for human parts.
Making the majority of the antibody molecule human to then go into humans.
Swapping out C domains.
CDR Grafting.

Fully Human Antibodies Creation

In 1988 and this kind of antibody was made by in vitro properties by phage display which we'll look at in a moment.
A mouse in which the mouse immunoglobulin variable region genes were replaced by human variable region genes at both the heavy chain and the light chain.
Actually starting with just human B cells.

CDR3 Grafting

The DNA encoding the CDRs are reconstituted from the mouse V genes and used to replace the CDRs in the human.
The are two very, very important examples here. Herceptin is used extensively in in breast cancer. I think that's trastuzumab as well. Yep So these are extremely important clinical antibodies and they've been developed in this process.

Generating Monoclonal Antibodies

How do you isolate individual B cells from a vast number and find that they're specific for the thing that you're interested in?
How will you keep them producing antibody forever?
Answer: fusing the population of antigen-reactive B cells with an immortal cell line (transformed or cancer cell line).
Making a fusion transfers the growth properties of the immortal cell line onto the antigen-specific B cell.
Separating out the fusion so that every fused cell pair of B cell and immortal cell is growing separately from all of the other events that have happened.

Process

Challenge a mouse with antigen. Activate B cells tha recognize that antigen and begin to proliferate.
Extract activated B calls. Grow them in isolation to allow each of them to make a single species of antibody recognizes an aspect of the antigen at the outset.

Fusion Technology

Fuse the spleen cells from a mouse that had been immunized with an antigen, so it was undergoing an immune response to sheep red blood cells in this case, and they fused it with a mouse myeloma.
-genius of Kohler and Milstein was to realize that if they forced that cell line, the myeloma, to fuse its nucleus with a B cell from an immunized mouse then the myeloma would retain the chromosomes for the immunoglobulin heavy and light chains from the B cell but also keep the genes it needed for continuous growth and antibody production that it had initially as a myeloma.

Process modified

Recover spleen and attempt to use all of the spleen cells with myeloma cells (an antibody secreting cells which grow forever in a dish). Fusion results that bind to our initial antigen can be selected.
Myeloma cell modification: does not make it's own antibodies, it has no genes that encoded immunoglobulin genes, the immunoglobulin proteins.
further innovations have been made this process to make it more efficient. And one of them is to make the myeloma cells defective in production of nucleotides that they need for DNA synthesis.
so in this way only fusions can grow and only fusions with B cells can produce antibody.

End result

Allows fusions between B cells and myeloma cells.
From this process one can recover the immunoglobulin genes from the fusion that originate from the reactive B cell and from that recover the specificity of the antibody.

Uses for monoclonal antibody

Whatever you can think of.

Therapeutics

Used in cancer
Used for treatment of infections.

Diagnostics

Radio immune assays and ELISAs for measuring circulating hormones, blood groups, for HLA typing for pregnancy, for cancer, antigen detection for infection.

Manipulation

Used to supress immune function by blocking immune activation, which can interfere and promote interactions.