IMMUNOLOGY LECTURE 5: Immunoglobulin Structure and Antibody-Antigen Interactions - Study Notes

Page 2: Antibody structure and antigen recognition

  • Reading and format context: Practice quiz forthcoming (format: multiple choice). Reading assignment: Immunobiology textbook, Chapter 4: “Antigen recognition by B cell and T cell receptors”.

  • Immunoglobulin structure components:

    • Subunit structure: heavy and light chains

    • Immunoglobulin fragments and their uses: Fc, Fab, and F(ab’)2

  • Antigenic determinants of immunoglobulins:

    • Isotypes and subclasses

    • Idiotypes

    • Allotypes

Page 3: Terminology

  • Antigen (Ag) is recognized by T cell receptors (TcR) or B cell receptors (BcR).

  • B cell surface antibody is BcR. B cells originate from the Bursa of Fabricius in birds; equivalent in mammals is bone marrow.

  • T cells originate from the Thymus.

  • Historical notes on antibodies:

    • Initially found in the gamma fraction of electrophoretic separation; described as globular proteins called gamma globulins.

    • When functioning as antibodies, they are called immunoglobulins (Ig).

  • Analytical notes:

    • Electrical separation depends on net charge.

    • Densitometry scan used to locate where antibodies are found.

Page 4: Basic structure of an antibody/immunoglobulin

  • Overall architecture:

    • Bilateral symmetry

    • Two antigen-binding sites

    • Variable regions (N-terminus): VH and VL

    • Constant regions: CH1, CH2, CH3 and CL

    • Hinge region allowing flexibility

    • Disulfide bonds linking heavy and light chains; interchain and intrachain SS bonds

    • Carbohydrate moieties present on the molecule

  • Key regions:

    • N terminus: variable region

    • C terminus: constant region

    • Ends of immunoglobulin domains defined by disulfide bonds

  • Abinding site composition: formed by VH + VL pairing; contributes to antigen recognition

Page 5: Size and assembly

  • Molecular weights:

    • Light chain ~ 25 \,\mathrm{kDa}

    • Heavy chain ~ 50 \,\mathrm{kDa}

    • Full Ig molecule (2 heavy + 2 light chains) ~ 150 \,\mathrm{kDa}

  • Structural properties:

    • Bilateral symmetry

    • Interchain bonds; heavy and light chains held together by disulfide bonds; heavy-heavy and heavy-light links via SS bonds

    • Intrachain disulfide bonds

    • Disulfide bonds help define the ends of immunoglobulin domains

  • Implication: the canonical structure features a dimer of two heavy and two light chains with defined domain boundaries

Page 6: Immunoglobulin domains

  • Domain composition:

    • Each heavy (H) and light (L) chain is composed of individual immunoglobulin-like domains.

    • These domains are delineated by cysteines at the domain ends that form SS bonds (SH groups).

  • Broader relevance:

    • Immunoglobulin domains are common across other proteins and are defined by similar cysteine-mediated SS bonds.

  • Family relationship:

    • Immunoglobulin supergene family, which includes TcR, CD4, CD8, MHC class I and II molecules

Page 7: Gene amplification and deletions (genetic mechanisms)

  • Concept: Potential source of amplified genetic material that can produce deletions and duplications in immunoglobulin gene regions

Page 8: Consequences of gene duplications/deletions

  • Phenotypic outcomes:

    • Excessive duplication or deletion can be lethal

    • Smaller-scale deletions/duplications can be survivable and allow mutation of the second copy to acquire new function

  • Example motif: ABBA-type gene structure observed in the MHC region, potentially illustrating this mechanism

Page 9: Proteolytic fragmentation of antibodies

  • Proteases and fragments:

    • Papain (from papaya): cuts above the hinge region producing Fab (monovalent antigen-binding fragment) and Fc (Fragment crystallizable)

    • Fab is monovalent; Fc mediates Fc receptor (FcR) binding

    • Pepsin: cuts below the hinge yielding a main fragment F(ab’)2 (bivalent)

  • Key differences: Fab vs F(ab’)2

    • Valency: Fab is monovalent; F(ab’)2 is divalent (bivalent)

    • Length/structure considerations involve the heavy chain portion that remains in each fragment

Page 10: Effects of Fab vs F(ab’)2 on Ab-Ag systems

  • Practice prompts:

    • Predict the effect of adding Fab fragments to an antibody-antigen system capable of forming immune complexes

    • Predict the effect of adding F(ab’)2 fragments to the same system

    • Q: What happens if you add free Fab to the mixture? Does it matter whether Fab is added at the beginning or after precipitation has occurred?

  • Conceptual expectations (based on fragment properties):

    • Fab fragments compete with intact antibodies for antigen binding; monovalent binding can inhibit cross-linking and immune complex formation

    • F(ab’)2 fragments, being bivalent, can cross-link antigens and promote or stabilize immune complexes differently than Fab

    • The timing of Fab addition can influence the kinetics and extent of precipitation or complex formation

Page 11: Kabat and Wu; sequencing Bence Jones proteins

  • Bence Jones proteins: monoclonal light chains found in urine of patients with B cell tumors (neoplastic plasma cells; myeloma)

  • Kabat and Wu work: sequenced the VL domains of several cancer/myeloma patient samples

  • Output: assembled a variability plot from VL gene sequences to study diversity

Page 12: Hypervariable regions within framework residues

  • Finding: three hypervariable regions identified among framework residues in antibody sequences

  • These hypervariable regions contribute to antigen-binding diversity

Page 13: CDRs and hypervariable regions (Figure reference)

  • Figure 4.7 (part 3 of 3) shows the arrangement of CDRs within the variable regions

  • Key labeling:

    • HV1 (CDR1)

    • HV2 (CDR2)

    • HV3 (CDR3)

  • Concept: The immunoglobulin antigen-binding site comprises these hypervariable loops that interact with antigen

Page 14: Designing recombinant “humanized” antibodies

  • Design workflow:

    • Sequence the original antibody (often mouse-derived monoclonal)

    • Use software to identify CDRs (complementarity-determining regions) and surrounding framework residues that influence folding

    • Replace CDRs with human sequences while preserving key framework residues for proper folding

    • Replace constant regions with human sequences and optimize codon usage for expression systems

  • Expression considerations:

    • Often use Chinese Hamster Ovary (CHO) cells to maintain typical glycosylation patterns; other cell lines include NS0, Sp2/0 (mouse myeloma), HEK293, PER.C6

  • Flexibility in design:

    • Constant regions can be modified to suit intended use, including stability and durability

Page 15: Antibody isotypes and J chain

  • Five mammalian antibody isotypes: IgM, IgG, IgA, IgD, IgE

  • J chain presence across isotypes (simplified mapping):

    • IgM: J chain present (yes)

    • IgG: J chain absent (no)

    • IgA: J chain present (yes)

    • IgD: J chain absent (no)

    • IgE: J chain absent (no)

  • Related notes:

    • J chain contributes to polymerization in IgM (pentamer) and IgA (dimer) forms

Page 16: Immunoglobulin heavy chains and light chains; additional antibody structure concepts

  • Heavy chains and their isotypes:

    • IgM: mu (μ)

    • IgG: gamma (γ)

    • IgA: alpha (α)

    • IgD: delta (δ)

    • IgE: epsilon (ε)

  • Light chains:

    • kappa (κ)

    • lambda (λ)

  • Beyond isotypes:

    • Idiotype: the antigenic character of the antigen-binding site

    • Allotype: allelic variants encoded by alleles of the Ig heavy and light chain genes; inherited

    • Anti-idiotypic antibodies are possible (Jerne network hypothesis)

    • Anti-isotype antibodies can be produced

Page 17: T cell receptor (TcR) variability

  • TcR also contains variable regions capable of antigen interaction

  • However, TcR interactions are not typically with soluble antigen; they recognize peptide antigens presented by MHC molecules on the surface of cells

Page 18: Affinity, avidity, and the forces of binding

  • Five contributing forces to antibody-antigen binding

    • Individually weak; collectively yield high affinity interactions

  • Affinity is quantified by the association constant KA:

    • K_A = \frac{[Ab!!!Ag]}{[Ab][Ag]}

    • Note: The expression above is the association constant for monovalent binding (Fab)

  • Avidity: bivalent antibodies exhibit much higher apparent affinity due to multivalent binding

  • Law of mass action underpins these relationships

Page 19: Measuring affinity and practical approaches

  • Common measurement methods:

    • Equilibrium dialysis

    • Surface plasmon resonance (SPR)

  • Conceptual measurement workflow (example for KA):

    • Establish equilibrium between free antibody, free antigen, and complex (AbAg)

    • Measure amounts of free antigen on each side and total antigen to derive bound antigen

    • Determine free antibody concentration from starting amounts minus bound

    • Compute KA from the measured concentrations

  • Typical affinity ranges: approximately 10^{-9} \text{ to } 10^{-12} (M) for Ab/Ag interactions

Page 20: Why knowing affinity and avidity matters

  • Value of affinity/avidity data:

    • Enables comparison of different antibodies with the same specificity to identify the best candidate for a given application

  • Other important properties to consider:

    • Antigen specificity

    • Antibody stability

    • Antibody class/isotype (defines biological activities and effector functions)