Monoclonal Antibodies - Mechanism & Production

What Are Monoclonal Antibodies?

Historical Origin

Step-by-Step Production: The Hybridoma Method

1. Immunization

2. Spleen Cell Harvest

3. Cell Fusion with Myeloma Cells

• Not secrete their own antibody
• Lack the enzyme HGPRT (hypoxanthine-guanine phosphoribosyltransferase)

4. HAT Medium Selection

• Unfused myeloma cells die - they lack HGPRT, so the aminopterin blocks their only alternative nucleotide synthesis pathway
• Unfused spleen cells die naturally within a few days
• Only hybridomas survive - they inherit HGPRT from the spleen cell and immortality from the myeloma cell

5. Screening and Cloning

6. Bulk Production

Mechanism of Action

From Mouse to Human: Engineering Strategies

Modern Production Methods (Beyond Hybridomas)

1. Phage display - Combinatorial libraries of antibody variable (V) genes are expressed on the surface of bacteriophages and screened against purified antigen. This eliminates the need for animal immunization and generates fully human antibodies. Greg Winter received the Nobel Prize in Chemistry (2018) for this concept. - Henry's Clinical Diagnosis
2. Transgenic mice - Mice engineered to carry human immunoglobulin gene loci produce fully human antibodies upon immunization.
3. ADLib system (DT40 cells) - An ex vivo method using gene conversion in DT40 chicken B cells treated with trichostatin A, allowing rapid generation of mAbs using antigen-conjugated magnetic beads for selection.
4. Recombinant expression - Once the V-gene sequences are known, antibody fragments (Fab, scFv) or full-length antibodies can be expressed in E. coli, yeast, or mammalian (CHO) cell lines at industrial scale.

Key Advantages of Monoclonal Antibodies

• Unlimited production of a homogeneous reagent with highly consistent affinity and specificity
• Can be produced against non-purified antigens - only the desired epitope-specific clone is expanded
• Enable analysis of molecules on an epitope-by-epitope basis
• Identify isoenzymes, subtypes, isotypes, and conformational changes in molecules
• Highly useful in immunoassays, diagnostic probes, and therapeutic agents

Limitations

• A single mAb recognizes only one epitope, so antigens with heterogeneous epitopes are harder to characterize with a single mAb
• Insufficient reactivity in precipitation/agglutination assays because network formation is weak with a single antibody
• Can be cross-reactive if different antigens share the same epitope (e.g., CA 19-9)
• Production remains expensive and technically demanding

Maturation of Lymphocytes

Part 1: B Cell Maturation

Phase 1 - Antigen-Independent (Bone Marrow)

Phase 2 - Antigen-Dependent (Peripheral Lymphoid Organs)

1. Antigen binds the BCR (surface IgM/IgD) - activates the B cell
2. T helper (TH) cell interaction via CD40-CD40L and cytokines (IL-4, IL-5, IL-6, etc.) drives proliferation in germinal centers
3. Somatic hypermutation introduces point mutations in V-region genes, increasing affinity - a process called affinity maturation
4. Class-switch recombination (isotype switching): intrachromosomal recombination between switch regions deletes intervening C-gene segments (e.g., Cμ→Cγ1 for IgG switch), allowing the same V-region to pair with a different constant region
5. Activated B cells differentiate into:
   • Plasma cells - terminally differentiated antibody secretion factories (secrete abundant cytoplasmic IgG)
   • Memory B cells - long-lived cells with refined specificity, poised to deliver a faster, stronger anamnestic response on re-encounter with antigen

Part 2: T Cell Maturation

The Thymus

• Cortex - densely packed with immature thymocytes + cortical epithelial cells; site of positive selection
• Medulla - medullary epithelial cells, dendritic cells, macrophages; site of negative selection; contains Hassall's corpuscles

Stages of T Cell Development

The Two Critical Selection Events

Positive Selection (Cortex)

• Occurs on cortical epithelial cells
• Thymocytes whose TCR binds self-MHC with low-to-intermediate affinity receive a survival signal and continue maturing
• Thymocytes that fail to recognize self-MHC at all → death by neglect (~95% of thymocytes)
• Result: ensures all surviving T cells are MHC-restricted (can interact with antigen-presenting cells)
• CD4 or CD8 lineage commitment also determined here: cells whose TCR binds MHC class II keep CD4; those binding MHC class I keep CD8

Negative Selection (Medulla)

• Occurs primarily on medullary epithelial cells and dendritic cells
• Thymocytes whose TCR binds self-MHC + self-peptide with high affinity → clonal deletion (apoptosis)
• Prevents self-reactive T cells from escaping to the periphery - central to self-tolerance
• AIRE (autoimmune regulator) gene in medullary epithelial cells drives ectopic expression of peripheral self-antigens (e.g., insulin, thyroglobulin), ensuring T cells specific for these antigens are deleted. AIRE knockout → autoimmunity (type 1 diabetes model)

Mature Peripheral T Cell Subsets

Part 3: NK Cell Maturation

Summary Comparison