📘 Fun & Detailed Theory Summary — Antibody Diversity, Somatic Hypermutation & Phage Display

1. Junctional Diversification 🔀 (VDJ Recombination Gets Messy!)

During antibody gene assembly, the immune system combines V, D, and J gene segments. But: when extra nucleotides are added at the junctions, the reading frame shifts, creating a different amino acid sequence → different antibody 🎯.

This randomness:

• Adds huge diversity to your antibody repertoire.
• Helps produce millions of unique antibodies.

👉 Key idea: Insertions at V–D–J borders drastically change CDR3 (the most variable region).

2. Somatic Hypermutation ⚡🧬

After a B cell produces its recombined antibody and displays it on its surface:

1. B cells with good binding to antigen get activated.
2. They move into germinal centers.
3. During proliferation, they undergo hypermutation specifically in the variable region (not the constant region!).
4. Mutations that improve binding result in that B cell expanding more.

Outcome: Affinity maturation → antibodies become stronger and more specific over time.

🧠 Important: Unlike normal DNA replication (very low error), B cells intentionally mutate their antibody genes in a controlled way.

3. Mimicking the Immune System in the Lab 🧪

We want to generate antibodies without relying on B cells, especially because:

• We can’t easily produce antibodies against self-antigens in vivo.
• We want antibodies against cancer or human proteins (the immune system avoids these to prevent autoimmunity).

So we engineer systems that mimic:

• Genetic variability
• Surface display of antibodies
• Selection for binders

This leads to…

4. Enter Phage Display 🦠✨ (George P. Smith’s Big Idea in 1985)

A filamentous bacteriophage (like M13) has:

• Thousands of copies of coat protein pVIII
• A few copies of pIII and pVI at the tip
• Genome = single-stranded DNA → historically used for Sanger sequencing

Smith realized: 👉 If you insert a foreign gene (e.g., an antibody fragment gene) into the phage genome at the position encoding protein 3 (pIII), the phage displays the fused protein on its surface.

So now:

• The genotype (inside) = antibody gene
• The phenotype (outside) = displayed antibody fragment

Exactly like a B-cell:

• Gene inside
• Antibody outside

This allows phase-display libraries:

• Billions of phage particles
• Each displaying a different antibody variant

🧪 Then we can select phages that bind a chosen antigen, just like selecting B cells in the immune system — but in vitro.

5. Why Do In-Vitro Antibody Libraries Matter? 🎯

Benefits:

1. Create antibodies to self-antigens, which immune systems normally avoid. → Useful for cancer, autoimmune markers, human proteins.
2. Controlled selection environments → We choose exactly what the antibody binds.
3. High diversity → Billions of variants without needing billions of mice.
4. Discovery tool → We can select antibodies without knowing the target beforehand.

6. Building the Antibody Library 🏗️

We use plasmids that include:

• Antibiotic resistance
• An origin for replication in bacteria
• A phage origin for packaging into phages
• The pIII gene
• A cloned variable antibody domain (VH)

But we only vary:

• CDR2
• CDR3 ← where most diversity naturally exists (encoded by V–D–J junction)

How do we introduce variability?

Options include:

• Error-prone PCR (low control)
• Ordering synthetic oligos with random nucleotides (better control)
• Trinucleotide synthesis 🧬 → Most advanced, because it lets you enforce:
  • Allowed amino acids
  • Natural-like frequencies
  • Avoid bad residues (e.g., Cys in CDRs)

Researchers study natural antibodies from databases (e.g., PubMed sequences) to determine:

• Which amino acids occur at which positions
• Their relative frequencies

Then design synthetic diversity that mimics nature.

7. Selections on Whole Cells 🧫 (Cell-Based Panning)

Cell membranes are extremely complex:

• Many proteins
• Different expression levels
• Carbohydrates, lipids
• Vary between cell types

Traditional immunization (injecting purified domains into mice) often fails because:

• Purified protein ≠ membrane-embedded protein
• Wrong structure or conformation
• Low-abundance cancer markers are overshadowed

Phage display solves this:

1. Present billions of antibodies to intact cells.
2. Only some bind.
3. Wash away non-binders.
4. Infect bacteria with the bound phages.
5. Amplify.
6. Repeat multiple rounds → enrich specific binders.

This was first demonstrated in 1993 by Greg Winter’s group, selecting antibodies to blood group antigens.

8. Challenges of Multiple Rounds of Selection 🔁

Repeated panning enriches "true" binders but also creates risks:

• Enrichment of background binders if they survive by chance
• Selection for phages that stick non-specifically
• Selection for binders to common surface molecules, not rare targets
• Loss of rare but highly specific antibodies
• Over-selection toward “sticky” antibodies

This is why careful experimental design matters:

• Negative selection
• Blocking
• Counter-selections
• Limiting rounds

🎉 Final Takeaway

Your immune system creates diversity through:

• VDJ recombination
• Junctional diversification
• Somatic hypermutation
• Affinity maturation

Phage display recreates these processes in vitro:

• Fuse antibody genes to pIII
• Display billions of variants
• Select for what binds your target

This enables:

• Therapeutic antibody discovery
• Targeting self-antigens
• Cancer diagnostics
• Studying membrane proteins in native conformation