Lecture 3: Antibodies & Gene Rearrangement

Origins and Definition of Adaptive Immunity

• Definition: Protective immunity that develops following an infection or vaccination. It is immunity that a person never possessed prior to exposure.

• Temporal Adaptability: The system changes over time to respond faster and more effectively to a pathogen.

• Immune Memory: This system can provide life-long protection against specific pathogens (e.g., measles).

• Massive Repertoire: A repertoire of billions of different naïve B and T lymphocytes is formed randomly before birth. These cells are essentially waiting for their specific "antigen" to be presented.

• Unique Specificity: Each and every lymphocyte has a unique antigen specificity generated through random processes.

• Diversity Mechanism: Diversity is caused by random gene rearrangement and recombination within the genetic loci coding for the B and T cell antigen receptors ($BCR$ and $TCR$).

• Affinity Maturation: The affinity of an antibody arising from a single $BCR$ increases over time and with the persistence of the antigen. The $BCR$ and $TCR$ gene loci are the only regions in the human genome capable of this rearrangement.

• Evolutionary Origins:     * First evidence appears in Agnathans (jawless fish), such as the Lamprey eel and Hagfish, which evolved approximately $300 \times 10^6$ years ago.     * These species use variable lymphocyte receptors ($VLR$), though they do not rearrange in the same way as $BCR$ or $TCR$.     * The mechanism for $Ig$ and $TCR$ rearrangement is almost identical to transposition in plants and bacteria.     * Transposition: A process where a gene changes its location within a genome. It requires two elements:         1. Transposase (recombinase): A specialist enzyme that cuts and repositions DNA bits. In humans, these are $RAG1$ and $RAG2$, active only in B and T cells.         2. Recognition Signal Sequences (RSS): Short conserved base pair sequences ($9$ or $7$ bp long) at the ends of gene segments. The transposase cuts at the $RSS$ and rejoins segments that may be many millions of base pairs ($Mbp$) apart.

Antibody Structure and the Immunoglobulin Fold

• General Composition: Antibodies are proteins produced by B cells, made of repeating $Ig$ domains.

• Structure Components:     * Two Heavy ($H$) chains paired by disulphide bonds at the hinge region.     * Light ($L$) chains paired to $H$ chains via disulphide bonds ($L-s-s-H-s-s-H-s-s-L$).     * Total molecular weight is approximately $150 \text{ kD}$.     * Arms: The "Y" shaped antibody has two identical antigen-binding sites at the tips of two flexible arms.     * Variable (V) Domain: Located at the N-terminal; contains the antigen-binding site.     * Constant (C) Domain: The invariant effector region ($CH2$ and $CH3$) where Fc receptors and complement component $C1$ bind. This defines the antibody's class and function.

• The Immunoglobulin (Ig) Protein Fold:     * Referred to as a $\beta$-barrel fold.     * Length is approximately $110$ amino acids.     * Structure consists of two anti-parallel $\beta$-pleated sheets (described as two "cupped hands").     * A stabilizing covalent disulphide bond connects the "palms," and loops join the "fingers."     * Flexibility: The loops are unconstrained, allowing amino acid sequences to vary greatly without disrupting the overall stability of the fold.     * Fold Details: $12.5 \text{ kD}$ $\beta$-barrel, $2$ anti-parallel $\beta$-pleated sheets joined by a central disulphide bond, with strands joined by $3$ loops.

The Antigen Binding Site

• Composition: Each antibody has two identical antigen-binding sites. Each site consists of $6$ loops in total: $3$ from the Light ($L$) chain and $3$ from the Heavy ($H$) chain.

• Hypervariable (HV) Regions: There are three hypervariable regions ($HV1$, $HV2$, and $HV3$) in the sequence where amino acids are seldom the same.

• Complementarity Determining Regions (CDR): The $HV$ loops are commonly known as $CDRs$.     * $CDR1$, $CDR2$, and $CDR3$ correspond to the folds in the protein that form the binding site.     * Variable Region Subdivisions: Framework regions ($FR1$, $FR2$, $FR3$, $FR4$) provide structure, while $HV$ regions ($HV1$, $HV2$, $HV3$) provide specificity.

Genetic Basis of Receptor Diversity

• Gene Locus Clusters: The $Ig$ $H$ and $L$ chain gene loci are divided into four clusters:     1. Variable (V): Approximately $100$ segments.     2. Diversity (D): Approximately $27$ segments.     3. Junctional (J): Approximately $6$ segments.     4. Constant (C).

• Rearrangement Steps in the H-chain Locus:     1. A randomly selected $D$ segment joins to a $J$ segment ($DJ$ joining).     2. A randomly selected $V$ segment joins to the new $DJ$ to form $VDJ$.     3. Intervening DNA (up to $100 \text{ Mbp}$) is discarded.

• Imprecise Joining: The joining of $D-J$ and $V-D$ segments is very imprecise. Random base changes occur before ligation, creating massive variation in the $CDR3$ loop (the $VDJ$ join).

• Significance of CDR3: The $CDR3$ loop sits in the center of the antigen-binding site and contributes most to the extreme hypervariability of antibodies and $TCRs$.

• Repertoire Scale: This process allows the production of more than $10^{11}$ different antibody molecules from only $30,000$ genes.

Clonal Selection and Affinity Maturation

• Lymph Node Dynamics: Development takes place in the lymph node follicle and germinal center.     * Follicle: A dense region with proliferating B cells, Antigen Presenting Cells ($APC$), and $T$ cells.

• Clonal Selection Theory (McFarlane Burnet):     1. A naïve $IgM$ B cell is triggered by its antigen.     2. The antigen drives clonal selection and expansion in the lymph node follicles.

• Affinity Maturation Process:     1. Somatic Hypermutation: Random mutations occur in the $Ig$ gene as B cells proliferate. Mutations leading to higher affinity clones allow those cells to bind available antigen more effectively.     2. Selection: Higher affinity clones expand faster, outcompeting slower-growing cells.     3. Differentiation: Mature B cell clones become antibody-secreting plasma cells, while others become long-lived memory cells.

• Temporal Progression:     * Primary Immunization: Antigen-specific $IgM$ appears, but protective immunity is poor.     * First Boost: High affinity $IgG$ appears due to class switching and affinity maturation. Protection improves.     * Second Boost: $IgG$ levels are high, and protection is stronger and longer-lived.

The Five Functional Classes of Antibodies (GAMED)

• IgM (μ gene):     * Default $Ig$ made by all naïve B cells.     * Membrane-bound form (monomer) serves as the B Cell Antigen Receptor ($BCR$).     * Blood form is a pentamer with $10$ identical binding sites, held together by a J chain.     * High avidity; excellent at activating complement component $C1$.

• IgG (γ gene):     * Most abundant class in the blood.     * Produced after switching from μ to γ gene.     * Neutralizes toxins and activates complement.     * Long-lived and can cross the placenta.     * Undergoes affinity maturation further to become very high affinity.

• IgA (α gene):     * Exists as a dimer in blood and mucosal secretions (breast milk, gut, tears, genitourinary tract).     * Provides passive gut immunity to neonates.

• IgE (ε gene):     * Least abundant in blood.     * Defends against parasites.     * Responsible for atopic allergies due to a high-affinity Fc receptor on Mast cells.

• IgD (δ gene):     * A form of $BCR$ found in membrane and blood forms.     * Crucial for B cell differentiation.

Affinity and Avidity

• Affinity: The sum of attractive molecular forces between two surfaces minus the repulsive forces.     * Metric: Higher affinity means fewer antibody molecules per unit volume are required to "find and bind" the antigen.

• Avidity: Results from multiple weak contacts (the "Velcro effect").     * The total binding force can be orders of magnitude higher than a single affinity interaction.     * Allows multivalent naïve receptors (like $IgM$) to bind pathogens before they have undergone affinity maturation.

Monoclonal Antibodies (mAbs)

• Discovery: A serendipitous discovery by César Milstein and George Kohler during research on affinity maturation at Cambridge University.

• Production Process:     1. Immunize a mouse with an antigen, followed by a boost.     2. Extract splenocytes (B cells from the spleen).     3. Fuse splenocytes with a mouse myeloma cell line using Polyethylene Glycol (PEG).     4. The result is a hybridoma: an immortalized fused B lymphocyte clone producing single antibodies.     5. Screen using ELISA.

• Pros of mAbs:     * High specificity; no "off-target" effects.     * Can be tailor-made to any antigen with specific affinity.     * Humanized versions can stay in the bloodstream for months.     * No toxicity; relatively cheap R&D costs.     * Can be modified to be "bi-specific" (arms targeting different things).

• Cons of mAbs:     * Mouse versions require engineering to look human.     * High manufacturing costs.

• Examples:     * Humira (adalimumab): Anti-$\text{TNF}α$ drug for inflammatory diseases. Projected global sales: $16 \times 10^9 \text{ dollars}$ in $2025$.     * Keytruda (pembrolizumab): Anti-$PD1$ immunotherapy for cancer. Projected global sales: $30 \times 10^9 \text{ dollars}$ in $2024$.     * Over $150$ FDA-approved therapeutic $mAbs$ currently exist.

• Historical Note: Cambridge University’s patent office did not see commercial value in the discovery and failed to patent it.