Structure and Function of Antibodies Folder Title: Antibody Filename: antibody(NoTP).ppt Updated: October 02, 2014 Folder: Antibody This section of the course will deal with the protein structure of antibodies, and will relate that structure to how antibodies carry out their two main functions. Those two functions are antigen binding, and inducing responses in the host after antigen recognition. The next section of the course will deal with how these diverse antibody structures are generated from a limited host genome. See parts of Chapter 3, Kuby Edition 7; B and T-Cell Receptors & Signaling Antibody Structure: pages 80 – 94

Targets and Weapons in the Specific Adaptive Immune Response Responds to Antigenic Determinants (Epitopes) Responds With: Lymphocyte Receptor Primary "Weapon" B-Cell Membrane-bound Antibody Extra-cellular Antibody T-Cell T-Cell Receptor Extra-cellular Cytokines Folder: Antibody As shown on this table, the primary "weapon" employed by the humoral B-cell-mediated specific adaptive immune response is soluble antibody. Antibody bound to antigen can then trigger additional responses such as activation of the complement cascade that will lyse labelled foreign target cells. Antibody labelling of target antigens can also attract the innate natural immune response to phagocytose and clear labelled foreign target cells. Thus there are a number of "secondary weapons" that the humoral immune response can utilize.

Folder: Antibody There are three questions to ask when considering antibodies as proteins. After dealing with antibodies as proteins, the genetic question remains about how these antibody proteins are coded for and generated.

Antibody Protein as the “Punter” Hands on the End of Arms = Recognize and Grasp the Ball Foot = Kicks the Ball Fab = Bivalent antigen recognition (Two “hands” needed) Fc = Effector function or business end Need both! Folder: Antibody The two functions of antibodies presented on the first slide are carried out by two different parts of the antibody molecule. It becomes necessary to determine how antibodies look in terms of their protein structure and how this structure relates to the functions of antibodies.

Fc Region Fab Region “Fc” = Fragment Crystallizable” “Fab” = Fragment Antibody Binding (not crystallizable)

Protein Structure of Antibodies A dimeric protein Heavy and Light Chains 2 Heavy – 2 Light (For IgG, 150K MW Antibody) or Multiple Sets (e.g.: 4H + 4L) for IgA or 10H + 10L for IgM (Macroglobulin; 900K MW) Different antibody isotypes and antibody roles IgG, IgA, IgM, IgE, IgD

Antibodies as Serum Proteins: Which serum proteins are they? What do they look like?

See Box 3-1, Figure 1 Kuby 7th Edition Adding Soluble Antigen to Blood Serum Which protein fraction decreased? Where did it go? Untreated Blood Serum Folder: Antibody Antibody activity is found in the cell-free liquid of the blood (the blood serum), and also elsewhere in the host. Antibodies in the blood serum are called serum antibodies, and blood serum with antibodies in it is called "antiserum". The serum antibodies are found mostly in the gamma globulin fraction of proteins in the blood serum. When one adds a specific antigen to an antiserum, it is the gamma globulin fraction that is depleted by antigen binding to antibody and subsequent precipitation of antigen-antibody complex. Antigen Added

Tetrameric Structure of IgG See Figure 4-7 Kuby, 6th Edition p. 86 Folder: Antibody The earliest studied and most well-characterized type of antibody, called "Immunoglobulin G" or "IgG" is a tetramer comprised of two light chains and two heavy chains, and has two Fab regions per antibody molecule. The H and Lsubunits are held together covalently by SS bridges between cysteine residues in the L and H chains, or between the two H chains. The two Fab regions appear like the arms of a "Y", while the other fraction of the IgG molecule, the Fc fraction is the leg of the "Y". The key question is: how does specific antigen recognition come about in the Fab regions of different antibody proteins? Figure 5-2, Kuby 3rd Ed. IgG4mer

Papain Digestion

Antibody – Antigen Binding Complementarity of Antibody – Antigen Binding

Antigen - Antibody Binding Antibody Light Chain Variable Region Fig. 4-6a Kuby 3rd Ed Folder Title: Antigens, AgAbKiss When the macromolecular antigen and the antibody that recognizes the antigen are in contact with another, their precise complementarity make them look like one single macromolecular structure rather than two distinct molecules. AgAb Kiss Antibody Heavy Chain Variable Region Influenza Virus Antigen

Antibodies as Proteins How Do Antibodies Bind to Virtually an Infinite Number of Different Possible Antigens? Proteins are not amorphous polymers. Proteins are not promiscuous in what they bind to. Start Here: Tuesday, September 23, 2014

Representation of Sequence Comparisons Among Light Chains from Antibodies with Three Different Antigen Specificities H3N-Ser-Val-Ile-Thr-Gly-Gly-Tyr-Ala... Thr-Glu-Ala-Val-Tyr-Ser-Met-COO- H3N-Ser-Ile-Met-Thr-Arg-Leu-Tyr-Gly..Thr-Glu-Ala-Val-Tyr-Ser-Met-COO- H3N-Thr-Gly-Gly-Thr-Lys-Leu-Tyr-Ile..Thr-Glu-Ala-Val-Tyr-Ser-Met-COO- Variable Amino Terminal Half Conserved Carboxyl Terminal Half (Positions 1 to 107) (Positions 108 - 214) This shows the first 8 Amino acids from the amino terminal V-region Folder: Antibody Comparison of amino acid sequences of antibody light chains with different antigen-recognizing specificities shows that there are positions of amino acid differences from antibody to antibody, and that these are concentrated in the amino-terminal half of the antibody light chain. In the schematic shown here, the positions where different amino acids appear in three different chains are shown in red, green, and blue. Where the same amino acid appears in all three chains that amino acid is shown in red. These sequence comparisons were first made in the late 1950’s and early 1960’s using “Bence-Jones proteins. Bence-Jones proteins are excreted in large amounts in the urine of patients with multiple myeloma and could be crystallized, purified, and sequenced in the early days of protein structure work. It turned out the the Bence Jones proteins were the light chains of an antibody generated by the unique clone of malignantB-cells that was the source of the myeloma in each different patient. This shows the last 7 amino acids from the carboxyl terminal conserved end.

The Following Two Slides Show the Variable Amino Acid Positions in the Amino Terminal One-half of the light Chains (107 Residues) And in the Amino Terminal One-fourth of the Heavy Chains (about 110 residues) The Heavy chain of antibodies is twice as long as the light chains: 440 Amino acids in each of the two heavy chains 220 Amino acids in each of the two light chains

Complementarity-determining Regions (CDR’s) from Light and Heavy Chains Come together in 3 dimensions to give the antigen-recognizing site.

Ag Binding Site (Fab) Ag Binding Site (Fab) Confers Biological Activity (Kicks the Football) (Fc)

Folder: Antibody The two Fab regions of an IgG molecule are shown binding to antigenic determinants (epitopes) on the surface of a virus. Note that the other part of the IgG molecule, the Fc region is hanging off "in space", not bound directly to the virus surface.

Following are Turning Point short answer questions. Please put all notes on the floor. Do not have any electronic devices other than your NXT transmitter. No consulting with other students. If you have a problem with your device, I can provide you with a loaner NXT device. If you have a problem using your NXT device, please ask Elisabeth for help. It is imperative that the integrity of these in-class Turning Point quizzes be maintained at the same level as we will do with the three written exams.

Antibody – Antigen Specificity? What Produces Antibody – Antigen Specificity? Why Do We get Specificity and Very Tight Affinity?

Figure 9.13 The Biology of Cancer (© Garland Science 2007) Blue shows topology of pocket in MDM2 protein that binds to p53 How proteins recognize each other topologically (3-dimensional surfaces) Yellow is p53 protein showing peptide domain sequence that binds to MDM2 Control Protein

Large surface protein epitope recognized Smaller low molecular size epitopes recognized

Small peptide antigen binding to an Fab (Fragment-antibody-binding) fragment of a complementary antibody

Conformational change in antibody upon binding antigen (“induced fit”) Light and heavy chain CDR regions move to better complement the antigen Peptide antigen from an HIV protein antigen Conformational change in antibody upon binding antigen (“induced fit”)

Movement of Peptide-binding Pocket Accompanying Antigen Binding: Fab Fragment of Antibody to Hemagglutinin Peptide Figure 5-11 Kuby, 3rd Ed AgAbMove

How does the antibody protein recognize its complementary antigen so precisely? Precise topological, spatial, and directional arrangement of stabilizing bonding. Minimization of repulsive interactions.

Immunoglobulin Isotypes Structures and Functions Start Here: Thursday, September 25th

See Figure 4-6, Kuby 6th Edition p. 85 Folder: Antibody The variable regions of the heavy and light chains are shown in red. The conserved regions are shown in gray. The conserved carboxyl terminal one-half of the antibody heavy chains constitute the Fc region of the antibody molecule. There are carbohydrate groups covalently attached, indicated as "CHO" in the diagram. There are five genetically different heavy chain types, designated mu, gamma, alpha, delta, and epsilon. These are the antibody isotypes that will be discussed later in this Folder. See Figure 4-6, Kuby 6th Edition p. 85

κ or λ forms See Figure 4-6, Kuby 6th Edition p. 85 Heavy Chain Variable Regions Including hypervariable CDR’s (Complementarity determining regions) κ or λ forms Folder: Antibody The variable regions of the heavy and light chains are shown in red. The conserved regions are shown in gray. The conserved carboxyl terminal one-half of the antibody heavy chains constitute the Fc region of the antibody molecule. There are carbohydrate groups covalently attached, indicated as "CHO" in the diagram. There are five genetically different heavy chain types, designated mu, gamma, alpha, delta, and epsilon. These are the antibody isotypes that will be discussed later in this Folder. See Figure 4-6, Kuby 6th Edition p. 85 Heavy Chain Iso-forms

The Heavy and light chains are labeled incorrectly in the Kuby Immunology Powerpoint slides. The figure is labeled correctly in the book.

Chain Structures of the five immunoglobulin classes in humans (adapted from Kuby, 2nd edition) Class Heavy Light Sub-Classes Subunit Formula Chain Chain IgG γ κ or λ γ1, γ2, γ3, γ4 γ2κ2 γ2λ2 IgA α κ or λ α1, α2 (α2κ2)n (α2λ2)n n =1,2,3,4 IgM μ κ or λ None (μ2κ2)n (μ2λ2)n n = 1 or 5 IgD δ κ or λ None δ2κ2 δ2λ2 IgE ε κ or λ None ε2κ2 ε2λ2 Folder: Antibody This table shows the five major isotypes of immunoglobulins, designated IgG, IgA, IgM (for "macroglobulin" because IgM is so large), IgD, and IgE. There are sub-classes of IgG and IgA with differences in the conserved region sequences of their heavy chains. IgG, IgD, and IgE have two heavy chains and two light chains. IgM and IgA have multiples of the H2, L2 motif.

Figure 5-17(a) Kuby, 3rd Ed. IgAModl Folder: Antibody The IgA dimer is held together by a small peptide called the"J" or joining peptide. IgA is secreted into the mucous fluids on the respiratory and digestive systems, so they are the first Ig's to contact exogenous antigens. IgA is by far the most prevalent Ig isotype. Figure 5-17(a) Kuby, 3rd Ed. IgAModl

Structures of Four Sub-types of IgG See Figure 4-18 Kuby, 6th Edition p. 98 IgGSubs Figure 5-16, Kuby, 3rd Ed.

Properties & Activities of Human Serum Immunoglobulins (from Table 4-2, Kuby Immunology, 4th Ed. p. 96) Property or Activity IgG* IgA** IgM IgE IgD# Mol Wt (KD) 150 150 – 600 900 190 150 H Chain gamma alpha mu epsilon delta Serum Conc (mg/ml) 0.5 – 9 0.5 - 3 1.5 0.0003 0.03 Serum Half-life (dys) 8 - 23 6 5 2.5 3 Activate Complement Yes No Strong No No Cross Placenta Yes No No No No Membrane (mIg) Form No No Yes*** No Yes Fc Binds Macrophages Yes No ? No No Mucosal Presence No Strong Yes No No Induces Mast Cell No No No Yes No * 4 Sub-classes IgG1, IgG2, IgG3, IgG4 **2 Sub-classes IgA1, IgA2 (exists as mono-, di-, tri, tetramer) *** mIgM is monomer. Serum IgM is pentamer # Has no known effector function. Is a membrane-bound antigen receptor Folder: Antibody This is a condensation of Table 4-2 in Kuby immunology, 4th edition, designed to make the information more manageable in class and when using the computer access. Key points are: (1) Most of the IgG sub-types can pass the maternal-fetal placental barrier. IgM and other isotypes cannot. (2) IgM is excellent at activating complement. IgG sub-types c an also activate complement. The other isotypes cannot. (3) IgA is the primary isotype that functions in the extra-cellular mucosal barriers of the gastro-intestinal and respiratory tracts. It is thus the first isotype to "see" approaching pathogens. (4) IgE is the allergy-related isotype that triggers Type I allergic hypersensitivity by cross-linking Fc receptors on tissue mast cells and making the mast cells degranulate to release bioactive factors. (5) IgM is very large compared to the others. It is the first isotype generated in B-cell differentaition and forms most of the membrane-bound Ig receptor that recognizes antigen and triggers a clonal response.(6) IgG is good at attracting the innate natural immune response to clear labelled antigens.

Effector Functions of Antibodies Functions of Fab Binding Neutralization or Blocking of Target Molecule or Particle Cross-linking and Agglutination of Target (Bivalent Fab Binding) Functions of Fc Region Complement Fixation and Lysis of Target Opsonization (Coating by Ab) and Phagocytosis of Target Targeting by Antibody for Cell-mediated Destruction (Antibody-dependent Cell-mediated Cytotoxicity: ADCC) Mast Cell Attraction and Activation by Bound IgE Immediate Type I Hypersensitivity, Type I Allergic Response, Localized and Systemic Anaphylaxsis Effector

Following are Turning Point short answer questions. Please put all notes on the floor. Do not have any electronic devices other than your NXT transmitter. No consulting with other students. If you have a problem with your device, I can provide you with a loaner NXT device. If you have a problem using your NXT device, please ask Elisabeth for help. It is imperative that the integrity of these in-class Turning Point quizzes be maintained at the same level as we will do with the three written exams.

Antibodies as Antigens Start Here Tuesday September 30, 2014 Why does this matters? If we want to use antibodies as therapeutic agents in patients, we have to understand and control the immunogenicity of the antibodies, or they will generate damaging and dangerous allergic responses, and be cleared from the patient and would be ineffective at best. Antibodies are not cells, so they don’t have transplantation antigens and they don’t have to be histocompatibility matched, but they have to go unrecognized as foreign proteins and they cannot be allowed to generate allergic reactions in the recipient. Start Here Tuesday September 30, 2014

Antibodies as Antigens Different Heavy Chain Isotypes (gamma, alpha, mu, epsilon, delta): Anti-isotype Antibodies (Anti-gamma, Anti-Alpha, Anti-Mu, etc) (Also differences in constant regions of kappa and lambda light chains) 2. Different individual mouse strains (or different people): Anti-allotype Antibodies (Antibodies from one person would raise anti-antibodies in a non-identical twin recipient) (1 and 2: Like any other proteins with multiple molecular forms) 3. Different antigen-recognition abilities: Anti-idiotype Antibodies. Anti-CDR’s for different antibodies Other proteins except for T-cell Receptors do not show these kinds of variations and are not immunogenic in this way

Transplantation Concepts and Nomenclature: For All Proteins From self, from identical twin, or from and to inbred animals of the same strain: Syngeneic, Isogeneic, Isologous From same species but not self or identical twin: Allogeneic (“allo” = other) From different species (e.g mouse to human) Xenogeneic (“Xeno” = foreign)

Transplantation Concepts and Nomenclature: Specifically for Antibodies Different Isotypes (IgG, IgM, etc.) in the Same Individual Isotypic Determinants The Same Isotype in Two Different Unrelated Individuals in the Same Species (CH1, CH2, CH3, and CL constant regions) And “Framework” Parts of VH and VL Allotypic Determinants (“allo” = other) The Same Isotype in Identical Twins or in Mice of Same Inbred Strain: Specific for Different Antigenic Determinants 6 Different CDR Regions or Idiotopes Idiotypic Determinants

Isoforms of the same antibody in the same individual

Allotypic determinants (different individuals) are in the Constant Regions of Heavy and Light Chains

Idiotypic determinants are in the V-regions of Heavy and Light Chains , especially at the CDR’s)

Humanized monoclonal Ab Therapeutic Monoclonal antibody would use antibody matched as isotype and matched in the framework allotype in V-regions (i.e matched as close as possible to the patient). Then graft in the CDR’s from mouse to get the antigenic specificity that is needed. Foreign idiotopes could go unrecognized and be non-immunogenic. Matched human isotype and human allotype. Graft in mouse CDR’s Humanized monoclonal Ab

Making Monoclonal Antibodies Now Immortal!

Following are two Turning Point short answer questions. Please put all notes on the floor. Do not have any electronic devices other than your NXT transmitter. No consulting with other students. If you have a problem with your device, I can provide you with a loaner NXT device. If you have a problem using your NXT device, please ask Elisabeth for help. It is imperative that the integrity of these in-class Turning Point quizzes be maintained at the same level as we will do with the three written exams.

View from OnLine at Textbook Web-site: Molecular Animation of Immunoglobulin Structure Molecular Animation of Cells of the Immune Response Molecular Visualization of Immunoglobulin Structure Molecular Visualization of Antigen-Epitope Interactions For OnLine Access to Immunology Edition 6 Information: http://bcs.whfreeman.com/immunology6e

Avastin for Breast Cancer: Possible withdrawal of FDA approval. Sept   http://www.cnn.com/video/#/video/health/2010/09/17/dnt.cohen.breast.cancer.cnn?iref=allsearch View Cancer Warrior, NOVA OnLine Video, 2001 http://www.pbs.org/wgbh/nova/body/cancer-warrior.html Judah Folkman, Endostatin, Angiostatin, and Targeting Cancer Vascularization “Judah will cure cancer in three years” (J. D., Watson, 2001) Anti-angiogenesis factor receptor monoclonal antibody: Avastin