Lecture 4- Antibodies Discovery of antibodies Specificity Variability Protein Structure of antibodies Antibody: Antigen interactions Antibody classes (isotypes) Monoclonal antibodies (Hybridomas)

Antibody structure and generation of B cell diversity Antibodies (Ab) are circulating proteins that specifically bind to foreign molecules--AKA immunoglobulins (Ig) Each antibody has a specificity different from the others Antibodies are made by B cells that have differentiated to become plasma cells Each B cell makes ONE and only ONE type of antibody--clonal selection Antigens are anything that is bound by an antibody Immunogens are antigens that elicit an antibody response. All immunogens are antigens, but the converse isn’t necessarily true.

Lymphocytes have unique, clonally distributed antigen receptors B cells 13 14 15 16 17 18 19 20 2 3 4 5 6 7 8 9 1 10 11 12 10 Antibodies 10 10 10

Day 0 2 4 6 8 Resting B cell Antibody forming cell (plasma cell) Y

Brief history of antibodies -In 1890 by von Behring and Kitasato described an activity in serum of toxin-immunized animals that neutralized toxin. Transfer of immune serum could protect naïve animals from diphtheria or tetanus. -Bordet found in 1899 that animals could make antibodies against erythrocytes of other species and that these could direct destruction of the cells along with serum “complement”. -1901-1920 Landsteiner demonstrates the ABO blood group system (Rh in 1940). -1930s Heidelberger Quantitative precipitin reactions -1930s Landsteiner’s analysis of antibody specificity -1960s Edelman, Porter, and Hilschmann’s elucidation of the primary and secondary structure of Abs. Discovery that Bence-Jones proteins were immunoglobulin L-chains. -1975 Kohler and Milstein invent monoclonal antibody technology -1976 Tonegawa clones first antibody gene

Early in vitro assays of antibody activity The the end of the 19th century three assays were developed that could measure antibodies: 1) bacteriolysis--fresh serum from immunized individuals, which contain both antibody and the complement system proteins, could directly lyse bacteria in vitro. 2) precipitin reaction- which involves the binding of antibody molecules to antigens that allow the development of large arrays that are poorly soluble. 3) agglutination- for example, erythrocytes of one species injected into another provoke antibodies that can be detected by their ability to cause aggregation of the cells.

Antibody is often a major serum protein

Landsteiner and the age of haptens Antibodies can be quite specific and can be raised to synthetic compounds m-azobenzenesulfonate

1) How does the antibody system manage to be so specific? 2) There are many antigens, virtually all of which can be seen specifically by antibodies. If all of these natural antigens, and even non-natural compounds, can be seen by the antibodies, there must be a huge number of different potential antibodies. How can they be encoded in DNA?

Hilschmann and Craig’s light chain sequencing data variable constant Strategy: to sequence antibody light chain proteins from patients with Bence-Jones proteins. These are monoclonal antibody L chains secreted into the urine of patients harboring a myeloma (plasma cell tumor).

How variable? Millions of different variable regions ~10 different constant regions How constant?

IgG immunoglobulin G First antibody class discovered, it represents ~80% of antibodies in the blood Fab fragment antigen binding Fc fragment crystallizable

Antibody structure (IgG) Figure 2.2 Antibody structure (IgG)

Figure 2.3 “Fab” fragment antigen binding Fc fragment crystallizable

Effector/ triage function (recruits innate immune cells) Information/ Specificity Effector/ triage function (recruits innate immune cells)

Illustration of the flexible hinge of antibodies Illustration of the flexible hinge of antibodies. Antibody in blue, divalent hapten in red.

Antibody flexibility

Ig structure.2

IgG complete structure

Entire Ig structure

Fig 2.6 V and C domains

Ig superfamily

Light chain CDRs contribute to part of the combining site Fig 2.7

Differences between antibodies are concentrated in hypervariable loop regions of V regions

The basis of antibody binding Depending on the nature of the antigen: Hydrophobic interactions Van der Waals forces Electrostatic interactions Hydrogen bonds Non-covalent, therefore reversible, binding Equilibrium affinity Ka= [Ag:Ab] [Agfree][Abfree]

Example Y Y + Ab* Y Y Y Y Y Y Y r/c r Bound Ab [Ab incubated] Given independence of binding sites the following relation applies: r/c = Kn - Kr and plotting our experimentally derived values of r/c vs. r allows the determination of K and n: Bound Ab [Ab incubated] Slope = -Ka r/c At x-intercept r= # sites/target r = Ab molecules bound/target c = free Ab concentration n = number of sites/target [sites with bound ligand] K = [free ligand] [free sites] r Antibody of uniform affinity High and low affinity

Typical affinity range of antibodies 105 - 109 M-1 The affinity constant K is related to the free energy of binding as follows: DGo = RT ln(Ka) where R is the gas constant (1.987 cal/mole-deg.), T is the absolute temperature, and ln(Ka) is the natural logarithm of the association constant. Thus a two-fold increase in binding energy translates to an affinity increase from Ka to (Ka)2.

The concept of epitopes, parts of antigens bound by antibodies Valency Fig 2.9

epitopes shown in white Polio virus epitopes shown in white VP1 protein

Different types of binding to antigen

Quantitative Immunoprecipitation Constant amount of Ab Zone of equivalence Ab Precipitation Zone of Ag excess Zone of Ab excess [Ag]

Immunoprecipitation

Low affinity IgM class High affinity IgG class IgM

Human immunoglobulin Isotypes Figure 2.4 These are monomer forms as they appear when expressed as a B cell antigen receptor. When secreted, the structures can be quite different. IgM is a pentamer in serum, and IgA can be a dimer.

Immunoglobulin classes

Valency

Human Immunoglobulins

Antibody classes have distinct and overlapping functions

Different antibody isotypes are found in different parts of the body

Regular gene: (eukaryote) Reminder about gene structure and the central dogma of molecular biology RNA AAAA Regular gene: (eukaryote) promoter DNA Exon 1 Exon 2 Exon 3 RNA splicing AAA mRNA AAA Many genes generate alternative splicing isoforms Translation on ribosomes to protein Translation Protein product Alternative protein product

B cell antigen receptor Differential RNA splicing determines if an antibody is secreted or remains as a membrane receptor B cell antigen receptor is a membrane bound form of antibody B cell

Antibody gene One exon is assembled from separate pieces by DNA rearrangement in immature lymphocytes DNA V Naïve B cell DNA Antigen stimulated B cell C heavy DNA On the antibody H chain, other exons are swapped in by a distinct DNA rearrangement

Antibody genes and the problem of generating diversity Concepts and summary Antibodies are highly specific Can see virtually any type of molecule Highly variable Immunoglobulin domain is a conserved structure Antigen contact sites are in hypervariable loops Antibody: Antigen interactions are reversible and characterized by affinity There are multiple antibody heavy chain classes (isotypes) that determine anatomical distribution and function. Monoclonal antibodies (Hybridomas) are useful tools in biology and medicine. Next time: Antibody genes and the problem of generating diversity