(9/19) Hanakahi Lecture: Antibody Structure and Function Antibody Structure: Classic Ab is composed of 4 polypeptide chains, 2 identical heavy, 2 identical lights. ~150kDa (big)

- Significance: Being large, means it is pretty difficult to make biosimilars. - Fab domain: “Arms”. Supports Ag binding, comprised of both heavy and light chains - Fc Domain: “Stem”. Provides Fc receptor binding. Comprised only of Heavy chains. The constant domain! CH3

regions are held together by disulfide bonds. Post-translational modifications/carbs are incredibly important for the functionality of the Ab

- Hinge Region: Held together by disulfide bonds, confer flexibility. Makes cross-linking and bivalent bonding possible. Agglutination is possible when the Ab binds to 2 Ag at once

Immunoglobulin Isotypes: There are 5 different classes of immunoglobulins - Isotype is based on the heavy chain C domains (Constant region). g a µ d e (And also subclasses)

o Subclasses arise from difference in heavy chain sequences, or l k chains. - Besides the 5 types of heavy chains, there are two types of light chains - l k, which can match up with any heavy. - IgG: The gamma globulin is the predominant Ig in the blood, lymph, and also 15% of the total protein in serum.

o Structure: Two g H chains and Two k/l L chains. o Glycosylation: Carb modification may ultimately influence binding, immunogenicity, and

effector activity. The type of glycosylation depends on the environment where IgG develops o Features: Long half-life (23 days) à Good for passive immunization o Function: Agglutination, Opsonization. Fc region may stimulate NK cells. Activate complement

§ IgG can neutralize toxins through physical interaction with the immunogen o Agglutintion: For insoluble particles, based on bifunctionality (binding more than 1), this is a more

effective route of elimination, making the particle bigger facilitates elimination o Opsonization: Ab recognizes and binds to pathogen via Fab region, thus coating the pathogen surface.

With the Fc region exposed à phagocytic cell can bind to it. This can also occur with the complement system

- IgM: A pentameric (5 subunits) immunoglobulin o Structure: 5 subunits, each with 2 µ H chains and 2 L chains, joined by disulfide bonds,

exposing a special J chain polypeptide. There is an extra Ch domain in the µ H chain, which holds everything together. Has carbohydrate modifications similar to IgG

o Valence of 5: Though 5 Ig molecules, valence is not 10. The Y tips are much closer together, preventing only singlet binding each.

o Function: Very efficient agglutination (due to polyvalency), Activation of Complement (potent, only needs 1 IgM pentamer)

o Immune Response Kinetics: First there is production of IgM to steady state (day 10), IgG becomes more prevalent (day15?). On second exposure, at day 3 the IgG spike is huge! Secondary responses are principally mediated by IgG, requiring less time. à Vaccination works well

- IgA: The major Ab in external secretions (Saliva, mucus, sweat, gastric fluid, tears) o Structure: Monomer has 2 a chains and 2 k/l chains. Monomer: No known function

§ à Dimeric secretory form has 2 additional peptides, a Joining (J) chain similar to IgM. There is even a secretory component

o Function: Main defense against local infections. It does not activate the complement system, it is an efficient agglutinating Ab, multivalent (2 or 4?), and good against viruses

- IgD: Found mostly on the surface of B Cells o Function: Currently unknown, it is predicted to play a role in the elimination of B cells

that express self-reactive Ab - IgE:

o Structure: Similar to IgD but different carbohydrate modifications. Fc regions have high affinity to the receptors on mast cells (mucosa and connective tissue) and basophils (circulation)

o Function: Ag can crosslink bound IgE by binding to Fab regions. Crosslinking stimulates cells to degranulate and release Histamine and Heparin. Thus, IgE is associated with parasitic infections and mediating hypersensitivity/allergic reactions.

§ May also release arachidonic acid and its metabolites (prostaglandins, leukotrienes)

B Cell Biology – The Production of Antibodies- V(D)J Joining - Heavy Chain has both V and C. Light Chain has both V and C. Thus, both have a

variable and constant region, the variable come together to form the tips of the Y - LIGHT CHAIN: Only 1 gene coding for the C region - Kappa (k) Light Chain Gene Rearrangement: Recombinase performs complex gene

rearrangements. The VL region is coded by two separate gene segments, V and J. - Lambda (l) Light Chain Gene Rearrangement: On a separate chromosome than k,

has a similar process of gene rearrangement, transcription, splicing, translation. V and J - HEAVY CHAIN: Has multiple genes for C region. - Recombinase recognizes signature motif on Chr14, there is a similar sequence near Chr8 MYC oncogene

(responsible for driving proliferation). Accidental translocation may result in Leukemia - Variable Region: Vh, Dh, and Jh. Hence, V(D)J Joining. The D and J segments code for the CDR, which is a

hypervariable AA sequence. - Constant Region: V and J à Multiple genes! This is different from the Light chain - Antigen-Driven Affinity Maturation: Antigen stimulation causes clonal expansion and triggers processes to

chance the CDR of stimulated B Cells. à Somatic Hypermutation. Point mutations in the Ag-binding region. The resultant Ag-binding region can improve or undermine Ag affinity. Occurs between 1st and 2nd

o Enzyme: Cytidine deaminase converts Cytidine to Uridine à changes in AA sequence Isotype Switching: Different classes of Ab result from switching of heavy chain constant region genes

- Fc: The constant region of the heavy chain determines the isotype - IgM is synthesized first, upon recognizing an antigen (T Cell interaction)à change the flavor of Ab. The

Constant region is switched, but the Variable (VDJ) region (& entire Light chain) stays the same- recognize the same immunogen!

o Splicing joins the J region to µ or d via alternative splicing. Periodically, the S (Switch Region) is manipulated via alternative splicing to switch the Constant region.

- Location for Switch: Depends on the nature of the stimulus o (1): Ag binding à “We need defense, we need to switch” o (2): T Cell secreted Cytokines à Tells us what type of defense

- After Switching: The B Cell is committed to making that Ab, the region switched out is lost. Polyclonal Ab Production:

- (1) Inject Ag into rabbit (2) Ag activates B Cells (3) Plasma B Cells produce polyclonal Ab (4) Extract Antiserum o Sometimes do additional injections to “boost” Ag-driven affinity maturation. The more you “boost” the

more you will drive clonal selection and affinity maturation - Benefit of Polyclonal: 1000s of Abs each with different affinity, complex! – not reproducible, supply is limited by

the blood supply of the host - Overall cross-reactivity: Antisera may have Ab specific to completely different proteins unrelated to immune goal

Monoclonal Ab Production: - Method: (1) Inject mouse, and perhaps boost it a couple times. Within the Spleen, we have lymphocytes the are

clonally expanding, producing Ab for Ag. Extract spleen cells. (2) Take a cell culture called myeloma, a mutant cancerous cell line with the potential to divide indefinitely. (3) FUSION, recombining the genetic information, a merger of the two cells. (4) Select from the resulting culture the cells that are part-spleen/part-myelomaà Hybridoma. (5) Each monoclonal lineage are descendants of a single cell, producing the same monoclonal Abs

- Say What?: Monoclonal Ab (mAb) are produced in hybrid cells. These Hybridomas can be frozen, stored, and thawed out upon disease outbreak.

- Biosimilars: Here’s the issue when it comes to drug design and why this fairy tale doesn’t always have a happy ending. “Subtle changes in the chemistry of the mAb may induce large functional changes.” So modifications, such as glycosylation, which is always happening to the Ab, may introduce variation. So it will produce something that is not standardized, it will be similar, but not exact. We call them glycoforms.

Polyclonal Versus Monoclonal - Monoclonal are much more expensive to make than Polyclonal, largely because more training is required with

better technology and it takes more time. - The benefit of Monoclonal is that it will recognize 1 specific epitope on an Ag, rather than multiple like the

population of polyclonal. The Monoclonal have Less to No batch-to-batch variability. Used in cancer therapy