🧬 Agenda Overview

Main questions:

1. What’s the genetic background for antibodies?
2. How can we generate antibodies without animals?
3. What is phage display and how is it used as a discovery tool?
4. Case studies:
   • Identifying biomarkers of aging cells (focus on Vimentin)
   • Identifying functional antibodies for the brain 🧠

🧫 What Is an Antibody?

Antibodies are Y-shaped proteins made by B-cells that recognize and bind to specific antigens. Each antibody has:

• Variable regions 🌀 — determine antigen specificity
• Constant regions — structural support and immune function

🧩 V(D)J Recombination: Nature’s Diversity Engine

Antibody diversity arises from V(D)J recombination:

• V (Variable), D (Diversity), and J (Joining) segments combine randomly.
• Humans can generate around 65 × 27 × 6 = 10,530 different heavy-chain variable fragments!
• Further diversity from:
  • Junctional diversification (imprecise joining adds random nucleotides)
  • Somatic hypermutation (mutations during immune response)

→ Together, this creates a massive antibody repertoire capable of recognizing millions of antigens 💥

🔬 Single-Chain Fv (scFv) Fragments

• These are engineered minimal antibody fragments consisting of VH + VL regions connected by a short linker.
• Small but still bind antigens with high specificity!
• Because of their size, they can access hidden (“cryptic”) sites on proteins — perfect for studying tricky targets.

🧠 Think of them as the “nanobots” of immunology — tiny, precise, and powerful!

🦠 Mimicking the Immune System in a Test Tube

This is where phage display comes in.

🧬 Filamentous Phage: The Display Vehicle

A filamentous phage (bacteriophage) is used as a carrier to display antibody fragments on its surface.

Process:

1. Insert gene for scFv into the phage genome.
2. The phage expresses the antibody fragment on its surface.
3. Use these phages to “fish” for targets by binding them to antigens.
4. Phages that bind are collected and amplified — mimicking natural selection of B-cells, but in a lab! 🧪

🧫 Selecting Antibodies for Membrane Proteins

Challenge: Membrane proteins are hard to express properly in vitro.

• Using only ectodomains often gives misfolded proteins with incorrect epitopes.
• Protein conformation can differ between cell types.

💡 Solution: Perform phage selection directly on intact cells to ensure correct antigen structure!

🔁 Single vs Multiple Rounds of Selection

• Multiple rounds: favor high-affinity antibodies but may bias toward highly expressed antigens.
• Single round: preserves diversity (especially when followed by screening or next-gen sequencing).

⚠️ Problem: Background binding (non-specific phages). ✅ Solution: Protease-sensitive helper phage — degrades background binders and improves specificity.

🧪 First Cell Selections

Phage selection was tested on various human and murine cell lines:

• Human epithelial, embryonic kidney, embryonic lung fibroblast, osteosarcoma, neuroblastoma, etc. → Each line reveals different binding patterns, shown via ELISA.

Jensen et al., 2002 (Mol. Cell. Proteomics 2:61–65) demonstrated:

• Selected antibodies from normal keratinocytes bound to specific cellular proteins in various cell types.

🧬 Immunoprecipitation Results

Using phage-selected antibodies:

• Ab-B3 pulled down Plectin (≈4684 amino acids) 🧱
• Ab-12 pulled down Laminin-5 (a trimeric ECM protein with chains of 1806, 1193, and 1172 aa)

These were identified through immunoprecipitation + SDS-PAGE + mass spectrometry. Result: Discovery of new biomarkers of aging and disease.

🌙 Moonlighting Proteins

Vimentin — typically a cytoskeletal protein — was found to have extra functions (“moonlighting”):

• The LOB7 antibody binds an epitope involved in angiogenesis (formation of new blood vessels).
• Such discoveries show how phage display can reveal multifunctional, context-dependent proteins.

⚗️ Heterogeneity and Single-Cell Targeting

Because tissues and tumors are highly heterogeneous, bulk analysis can mask rare cell types. Innovative techniques like:

• Shadow stick or micro-aspiration allow targeting of single cells or subpopulations (e.g., brain pericytes).

🎯 Aim: Identify cell-type-specific antibodies for precision medicine.

🧠 The C3 Antibody Case Study

• Target: Pericytes in brain vasculature.
• Methods:
  • Immunohistochemistry (IHC) on retina:
    • C3 antibody staining
    • Co-staining with α-SMA (pericyte marker)
  • Immunoprecipitation + MS identified Fibronectin as the binding partner.
  • Functional tests:
    • Scratch wound assay 🩹 (cell migration)
    • Tube formation assay 🩸 (angiogenesis)

Outcome: Demonstrated the functional relevance of selected antibodies — not just binding but affecting biological processes!

🧭 Summary

Recombinant antibody technology = a versatile discovery and therapeutic tool.

• Enables identification of disease-related proteins in their native environments.
• Captures dynamic, cell-type-specific protein changes.
• Antibodies can also modulate pathways directly — offering therapeutic potential.

🧪 Broader Research Areas

🙌 Acknowledgements

Funding from:

• Danish Research Council, EU FP6/FP7, H2020
• Novo Nordisk, Lundbeck Foundation, Cancer Society, APM Foundation
• Collaborations with Bioneer A/S, Bio-Y, and others

Key contributors: Ole Aalund Mandrup, Mathias Jørgensen, Kim Bak Jensen, Karen Marie Sørensen, Theresa Meldgaard, Jesper Just.