Case Study - Monoclonal Antibodies

Question 1

What are Monoclonal Antibodies?

Answer 1

The body naturally produces antibodies, which are elements of the immune system produced by B-lymphocytes, that bind to foreign proteins in the body known as antigens, which the aim of eliminating them

Question 2

What are the types of mAbs

Answer 2

1. Murine monoclonal antibodies
2. Chimeric monoclonal antibodies
3. Humanized monoclonal antibodies
4. Human monoclonal antibodies

Question 3

What do proteases, lipases and amylases breakdown?

Answer 3

Proteases:
- Meat,
- Seafood,
- Soybean etc.

Lipases:
- Beef,
- foie gras,
- fresh cream,
- egg yolk,
- cheese etc

Amylases:
- chocolate,
- cake,
-biscuit,
- cookies,
- soft drinks
- alcohol

Question 4

Draw the different types of mAbs

Answer 4

Safwan’s lecture sliide 10

Question 5

What is an epitope?

Answer 5

It is part of an antigen that the host’s immune system recognises

Question 6

Epitopes in mAbs

Answer 6

Currently, the mAbs only target one epitope since they are from the same clone

Question 7

When was the first mAb produced?

Answer 7

1975

Question 8

When was Adalimumab (Humira): anti-TNFα for RA created?

Answer 8

2002

Question 9

What was Adalimumab (Humira): anti-TNFα for RA?

Answer 9

HUMIRA for RA is a prescription medicine used alone, with methotrexate or certain other medicines to reduce signs and symptoms of moderate to severe rheumatoid arthritis in adults

Question 10

How are mAbs produced?

Answer 10

The production of monoclonal antibodies is an in vitro process with the use of tissue-culture techniques.

1. Immunization of mice with cancer-specific antigens to stimulate antibody production
2. Isolation of antibody-secreting plasma cells
3. Fusion and generation of hybridomas
4. Selection in HAT medium and ELISA screening
5. Expansion of selected hybridoma to produce monoclonal antibodies

Question 11

Give a detailed process of how mAbs are produced

Answer 11

• Producing monoclonal antibodies (mAbs) is initially done by identifying a specific antigen, and immunizing an animal with the antigen multiple times. The most commonly used animal models are laboratory mice.
• The B-cells of the immunized animals are removed from the spleen and then fused with cancer B-cells known as the myeloma cells.
• The fusion of adjacent plasma membranes of the myeloma cells is done using polyethylene glycol, however, it has a low success rate, and therefore the selective medium must have the fusion activity as well to enhance cell growth.
• Myeloma cancer cells have an immortal characteristic of continuously proliferating, unlike the normal B cells which proliferate for a period of 6-8 hours, and they normally have lost the ability to synthesize hypoxanthine-guanine-phosphoribosyl transferase (HGPRT), an enzyme necessary for the degradative synthesis of nucleic acids.
• The myeloma cells are placed in a selective medium known as the Hypoxanthine Aminopetrin Thymidine (HAT)-which is made up of hypoxanthine, aminopterin, and thymidine, where it allows the growth and yield of fused hybridoma cells, and the infused myeloma cells do not grow and infused B-cells die off.
• Hybridoma cells have the ability to grow continuously in culture as they produce antibodies. They are then screened for the desired or specific monoclonal antibodies, and those producing the desired mAbs are then transferred and grown in tissue culture.
• Harvesting is done periodically and the monoclonal antibodies are then purified from the medium.
• Growing and harvesting of these monoclonal antibodies are done for several weeks in large media quantities in order to produce enough mAbs that can be used for experimentation or to treat at least a single patient.
• The monoclonal antibodies produced are in millions of numbers and they are specific for the antigen that was initially injected into the animal model.

Question 12

Functions and Applications of Monoclonal Antibodies

Answer 12

• Monoclonal antibodies are used in the treatment of several diseases and disorders and their application is known as immunotherapy.
• Some diseases and disorders treated using mAbs include: Cancers, Rheumatoid arthritis, Multiple sclerosis, Systemic Lupus erythematous, Cardiovascular diseases, Crohn’s disease, Ulcerative colitis, Psoriasis, and Rejections associated with transplantation
• Monoclonal antibodies are widely used in therapies, laboratory technique studies, and research for potential treatments for certain infections, disorders, and cancers
• Commonly, monoclonal antibodies were initially studied in cancer treatment where they are currently used in the treatment of some types of cancer.

Some of the specific applications include:

• Some monoclonal antibodies are designed to target specific tumor antigens. They have been used to stimulate the production of anti-idiotypic antibodies stimulating a strong antitumor immune response when they are inoculated in patients with B-cell lymphoma. However, anti-idiotypic antibodies are developed in animal models, which hiders production of monoclonal antibodies in humans. Though, humanized antiHer2 monoclonal antibodies Herceptin has proved effective in patients with chemotherapy-resistant breast cancer.
• Monoclonal antibodies are used to effectively bind the Tumor Necrotic Factor-alpha (TNF-alpha), which is a cytokine that helps in the progression of Rheumatoid arthritis (RA). Hence monoclonal antibodies are used as a therapeutic means for Rheumatoid Arthritis.
• Monoclonal antibodies have been generated against Tumor-specific Transplantation Antigens (TSTAs). These are antigens that result from gene mutations that cause altered proteins that are expressed by tumor cells. Practically, the patient tumor cells are tagged with monoclonal antibodies that have toxins or radioactive materials. This delivers a direct ‘magic-bullet’ therapeutic effect to the tumor and spares the healthy cells.
  They are used as an identification tool for several cancers and to also deliver drug therapies to target cancer cells and initiate immune responses against the cancer cells.
• Monoclonal antibodies are used in the diagnosis of several diseases by detecting specific antigens circulating in the body tissues and detecting them by the use of immunoassay techniques.
• Currently, monoclonal antibodies are being studied by the COVID-19 Prevention Network. for the treatment of COVID-19. Some trials have been rallied out in the US to understand the role of monoclonal antibodies in providing short-term protection against SARS-CoV-2 the causative agent of COVID-19.

Question 13

Draw the difference process of creating the different mAbs

Answer 13

Safwan’s lecture slide 13

Question 14

What are the uses of mAbs?

Answer 14

1. Identification of phenotypic markers unique to particular cell types.
2. The diagnosis of many infectious and systemic diseases relies on the detection of particular antigens or antibodies in the blood, urine, or tissues by the use of monoclonal antibodies in immunoassays.
3. Tumor identification.
4. Advances in medical research have led to the identification of cells and molecules that are involved in the pathogenesis of many diseases
5. Functional analysis of cell surface and secreted molecules

Question 15

Limitations of mAbs

Answer 15

Monoclonal antibodies are most easily produced by immunizing mice, but patients treated with mouse antibodies may make antibodies against the mouse Ig, called human anti-mouse antibody (HAMA). These anti-Ig antibodies block the function or enhance clearance of the injected monoclonal antibody and can also cause a disorder called serum sickness.

Genetic engineering techniques have been used to expand the usefulness of monoclonal antibodies. The complementary DNAs (cDNAs) that encode the polypeptide chains of a monoclonal antibody can be isolated from a hybridoma and these genes can be manipulated in vitro.

Fully human monoclonal antibodies are also in clinical use. These are derived using phage display methods or in mice with B cells expressing human Ig transgenes. Humanized antibodies are far less likely than mouse monoclonals to appear foreign in humans and to induce anti-antibody responses.

Question 16

Who is Sir Gregory Winter?

Answer 16

Sir Gregory Winter was a Nobel Prize winner for his therapeutic work on monoclonal antibodies.

He founded the company Cambridge Antibody Technology (CAT), their core focus was on antibody therapeutic and primarily using Phage Display and Ribosome Display technology. CAT was then later acquired by AstraZeneca for $1.4 billion.

He later created or cofounded the company Domantis which also focuses on therapeutic agents. This was late acquired by GSK for $454 million

Question 17

What is Multiple Sclerosis (MS)?

Answer 17

Multiple Sclerosis is when the immune system attacks the protective sheath (myelin) that covers nerve fibres and causes communication problems between your brain and the rest of your body. Eventually, the disease can cause permanent damage or deterioration of the nerve fibres.

Signs and symptoms of MS vary widely between patients and depend on the location and severity of nerve fibre damage in the central nervous system. Some people with severe MS may lose the ability to walk independently or ambulate at all. Other individuals may experience long periods of remission without any new symptoms depending on the type of MS they have.

Question 18

What is the relationship between MS and mAbs?

Answer 18

Monoclonal antibodies (MABs) are one of the preferred treatments for multiple sclerosis (MS) due to their target specificity and usually high efficacy. These have usually targeted the immune system, which plays a key role in the pathogenesis of MS, especially during the early inflammatory stage

In MS, MABs are able to more specifically neutralize key immune players that negatively impact the central nervous system. Because of their specificity, they tend to have few off target effects and less drug–drug interactions minimizing their side effects, which tend to arise from their downstream effects on the immune system and reactions to the drugs themselves, although this problem has been greatly reduced with the advent of humanized MABs

Question 19

Who was Jesse Gelsinger and what was his role in the future of gene therapy?

Answer 19

Jesse Gelsinger was the first person who publicly identifies as having died in a clinical trial for gene therapy after having a negative reaction to the injection.

He suffered from ornithine transcarbamylase deficiency, an X-linked genetic disease of the liver, the symptoms of which include an inability to metabolize ammonia – a byproduct of protein breakdown.

This event leads the field of gene therapy to collapse, where gene therapists were blacklisted.

Question 20

What are the challenges that gene therapy is facing?

Answer 20

One: Delays in approvals
- The FDA is placing closer scrutiny on gene therapy products to ensure that they don’t enter the market prematurely. Drug manufacturers may go through multiple rounds of FDA review or “cycles” prior to approval, which can delay patients’ access to these new treatments but also ensures appropriate safety and efficacy.
- Even after approval, it can be difficult to get these new therapies to the patients who need them. That may be due to manufacturing and distribution challenges as well as complexity in patient selection and administration. These therapies may need to be given in a hospital setting under medical supervision. On top of their complexity, there are a limited number of certified treatment centres that can offer them.

Two: Prohibitive upfront costs:
- So far, the therapies approved are extremely expensive – more than $2 million for a single dose or one-time treatment – and future treatments will likely command higher price tags. This is due to a long, complicated process for the development, manufacturing and delivery of these products. And they often target diseases with smaller patient populations. A higher price tag is used to recoup the cost of developing a drug for a limited market of patients.

Three: Clinical unknowns:
- Despite the curative potential of gene therapies, they’re still new approaches to treatment and could carry significant health risks. Safety and delayed adverse events associated with gene therapy have been a concern since the first clinical trials 20 years ago.
- Some of the risks include life-threatening immune responses, certain types of cancer, allergic reactions, or damage to organs or tissues
- It’s important to recognize the need to balance those risks and address outstanding questions about the durability of gene therapies. It’s still too early to tell if they’re truly “cures” or if the diseases will return. Even if results degrade over time, gene therapy may still be a better and more cost-efficient treatment option to improve the length and quality of life for patients with genetic disorders.

Question 21

Where does the cost come from for the treatments to be so expensive?

Answer 21

The cost comes from the Manufacturing of the drugs. As the capturing and polishing stages of the manufacture, you would lose a decent amount (%) of your product and would if these processes were done incorrectly, there is potential to lose even more product, therefore there is more requirement to manufacture more drugs.

Question 22

What is the future of gene therapy?

Answer 22

Gene therapy hopes to develop new genetic therapies for both common and rare diseases.

Their goal is also to tailor medicines for each person instead of the traditional “one-size fits all” medicine. These personalised medicines could help improve each patient depending on their needs and genetic coding.

Question 23

What does codon bias mean?

Answer 23

the phenomenon where specific codons are used more often than other synonymous codons during the translation of genes

Question 24

Why do we need upstream processing?

Answer 24

1. To improve quality of the product
2. To improve upon the yield of the product

Question 25

what are inclusion bodies?

Answer 25

Inclusion bodies (refractile bodies) are insoluble aggregates of partially folded heterologous products. Because of their dense nature, they are easily observed by dark-field microscopy.

When expressed at high levels, heterologous proteins overload the normal cellular protein-folding mechanisms.

Aggregation is largely driven through hydrophobic patches

Question 26

Advantages of inclusion bodies

Answer 26

• Resistant to proteolysis
• 90% of the protein can be the heterologous product
• Higher yields than soluble proteins.
• Low-speed centrifugation after homogenisation.

Question 27

What are the refold screens?

Answer 27

• High density of inclusion bodies allow them to sediment rapidly than cell debris
• Low speed centrifugation after homogenisation
• Inclusion bodies are then dissolved in denaturaturants (urea)
• Denaturant is removed through filtration and diafiltration

Fermentation –> Cell Harvest –> Incusion Body washing –> Solubilisation –> Refold

Question 28

What is the Cytomine workflow?

Answer 28

1. Sample loading
   - Cells and assay reagents are loaded into cyto-cartridge.
2. Picodroplet
   - Cells and assay reagents are encapsulated into picodroplets.
   - hundreds of thousands of cells can be encapsulated and processed in a single run.
3. Productivity screen
   - Picodroplets are incubated (37C).
   - Cells secrete products and due to encapsulation, the product produced by a single cell can be detected
4. Droplet sorting
   - Picodroplets which have the highest level of detectable products are selected
5. Single droplet dispensing
   - Selected picodroplet are dispensed into a well of a 96-well plate.
   - 5 images are taken pre-dispensing.
   - Heat-map of the 96-well plate produced.
6. Plate imaging
   - 96-well plates image at multiple times points using cell metric.
   - Colony formation can be tracked to the single cell stage.