JT- Cancer, Autoimmunity, Dementia

Question 1

Who were the key figures in the history of antibody discovery, and what were their contributions? (4)

Answer 1

Paul Ehrlich (1901)

• Proposed “magic bullets” for targeted bacterial infection treatments, including syphilis.

Emil Behring (1890)

• Developed the diphtheria antitoxin serum, proving passive immunization could protect against toxins.

César Milstein & Georges Köhler (1975)

• Created the hybridoma technique to produce monoclonal antibodies (mAbs).

Jim Allison (2018)

• Developed immune checkpoint inhibitors, revolutionizing cancer immunotherapy.

Question 2

What is the purpose of hybridoma technology?

Answer 2

To produce monoclonal antibodies by fusing B cells with myeloma cells.

Question 3

What are the key steps in hybridoma production (5)

Answer 3

1. Immunization – Mouse is injected with an antigen.
2. Myeloma cell selection – Uses cancer cells lacking HGPRT enzyme.
3. Fusion – Mouse B cells are fused with myeloma cells using PEG.
4. Selection – Hybridomas survive in HAT medium.
5. Screening & growth – Identifies hybridomas producing the desired antibody.

Question 4

Why do unfused myeloma and B cells die in HAT medium? (3)

Answer 4

• Unfused myeloma cells lack HGPRT and cannot grow in HAT.
• Unfused B cells have a limited lifespan in culture.
• Hybridomas survive because they combine B cell antibody production with myeloma immortality.

Question 5

What is a monoclonal antibody?

Answer 5

An antibody produced from a single hybridoma cell, binding to one specific target.

Question 6

What were the limitations of early murine monoclonal antibodies?

Answer 6

• Poor interaction with the human immune system.
• Recognized as foreign, leading to rapid clearance.
• Potential allergic reactions upon re-treatment.

Question 7

What are the main types of engineered antibodies? (3)

Answer 7

Chimeric antibodies

• Mouse variable domains + Human constant domains.

Humanized antibodies

• Mostly human, except for CDR regions.

Fully human antibodies

• Generated using transgenic mice or phage display.

Question 8

What were the key advancements in antibody engineering? (4)

Answer 8

Sherie Morrison (1984)

• Developed antibody gene expression in lymphoid cells.

Michael Neuberger & Terrence Rabbitts (1984)

• Created chimeric antibodies (rodent V regions + human C regions).

Greg Winter (1986)

• Developed humanized antibodies (only CDRs retained from rodents).

Campath-1H

• First successful humanized antibody therapy.

Question 9

What are the key steps in antibody engineering? (11)

Answer 9

1. Immunization – A mouse is injected with the target antigen.
2. Serum Analysis – Confirms antibody production (e.g., measuring IgG levels).
3. Extract B Cells – B cells are harvested from the spleen or lymph nodes.
4. Hybridoma Generation – B cells are fused with myeloma cells.
5. mRNA Isolation – mRNA is extracted from the B cells.
6. cDNA Synthesis – Converts mRNA into cDNA, which contains antibody genes.
7. Vh and Vl Amplification – The variable regions of heavy (Vh) and light (Vl) chains are amplified using PCR.
8. Recombinant Antibody Expression – Inserted into expression vectors (e.g., E. coli or mammalian cells).
9. Screening – Identifies clones producing the desired antibody.
10. Characterization – Affinity and stability are analyzed (e.g., ELISA, Biacore).
11. Antibody Engineering – Further modifications (e.g., humanization, affinity maturation).

Question 10

What is antibody phage display?

Answer 10

A technique that allows in vitro selection of antibodies with specificity and high affinity, without needing immunized animals.

Question 11

What are transgenic mice, and why are they important in antibody engineering?

Answer 11

Mice that have been genetically modified to produce fully human antibodies instead of mouse antibodies.

Question 12

What are the benefits of transgenic mice in antibody production? (3)

Answer 12

• Rapid generation of fully human, high-affinity antibodies.
• Reduced risk of allergic responses (no HAMA or HACA reactions).
• Eliminates the need for time-consuming genetic engineering.

Question 13

What company is known for using transgenic mouse technology in antibody production?

Answer 13

Genmab

Question 14

What are Single Domain Antibodies (dAbs), and what are their advantages? (3)

Answer 14

• Smaller antibodies consisting of only a single variable domain.
• Higher affinity compared to conventional antibodies.
• Better tissue penetration, making them useful for therapeutic applications.

Question 15

What are two major technologies for generating fully human antibodies? (2)

Answer 15

Phage Display

• In vitro selection of antibodies.

Transgenic Mice

• Mice engineered to produce human antibodies.