antibody structure and specificity discussion starts with page 105

1. Antibody Structure and Specificity(discussion starts with page 105) Specificity of antigen receptor determined before B-lymphocyte even encounters any antigen (while B-cell is still in the bone marrow) Receptor specificity is same as secreted antibody Somatic recombination in the bone marrow determines specificity of B-cell antigen receptor Uses germ-line (inherited) gene fragments (V,D,J & V,J) Generates amino acid composition of variable portions of heavy and light chains

3. The clinical process known as “desensitization” is an attempt to eliminate the “sensitized” mast cells and cause an isotype switch from IgE to any other isotype (e.g., IgG). Desensitization (sometimes called “allergy shots”) deliberately injects very small quantities of which one of the following materials into the allergic patient? A. antihistamines B. antibodies specific for the constant portion of all IgE isotype antibodies C. the allergen (the material to which the patient is allergic) D. antibodies that would bind to mast cells, initiating a Natural Killer (NK) cell-mediated destruction of any sensitized mast cells

4. With the exception of minor alterations in the nucleotide composition of variable regions as the result of somatic hypermutation, the specificity of a secreted antibody molecule of the IgG isotype is determined by which one of the following? A. isotype switching under the influence of T-helper cells B. somatic recombination involving random selection and assembly of gene fragments for the two variable portions of the antibody molecule C. the elimination of all B-lymphocytes specific for self peptides, leaving behind only B-cells specific for non-self antigens D. the short amino acid sequences at the carboxy-terminal ends of both of the receptor heavy chains that are embedded into a B-lymphocyte’s membrane – the secreted antibody has these same short sequences that are no longer inserted into any cell membrane (but serve to activate complement)

5. BONUS clicker question (correct answer worth one point) A. destruction of HPV-infected cells by primed T-cytotoxic cells B. destruction of virus-infected cells by Natural Killer (NK) cells C. prevention of attachment of the HPV to body cells by antibodies D. increased release of interferons (alpha and gamma) by HPV-infected cells

7. BONUS clicker question (correct answer worth one point) A. adds another way that complement can be activated B. increases the overall effectiveness of the vaccine C. overstimulates T-regulatory cells, resulting in a decrease in any possible autoimmune diseases the vaccine may cause D. reduces the release of any cytokines that could attract neutrophils to the area of the HPV virus infection, thus eliminating any possible involvement of toll-like receptors in the response

8. Athymic (nu/nu) “nude” mouse

9. A few more details about somatic recombination For each chain, the variable portion gene fragments and genes for constant portion are on same chromosome Light chains Kappa – two homologous chromosomes 2 (V,J & one kappa constant gene) Lambda - two homologous chromosomes 22 (V,J & 4-5 lambda constant genes) Heavy chains – two homologous chromosomes 14 (V,D,J & all five constant genes) Only one light chain chromosome and one heavy chain chromosome are eventually used by each B-lymphocyte (allelic exclusion) Many different gene fragments are available for use

10. “Control” of somatic recombination Recombinant signal sequences (RSS) flank each variable portion RSS separated either by 12 base pair “spacers” or 23 base pair “spacers”

11. “Control” of somatic recombination (continued) Recombinant activation genes (RAG-1 and RAG-2) One RAG binds to a 12-spacer, the other to a 23-spacer RAG-12 & RAG-23 spacers are brought together ALL nucleotides between the two gene fragments (including the RSS and any other gene fragments/RSS) are excised making a loop that is NOT used for anything The (now) contiguous gene fragments will be ligated Light chain – selection of one V + one J Heavy chain Selection of one D + one J -> DJ (done first) - must observe the 12/23 “rule” Then randomly selects one V and ligates it to DJ -> VDJ Video http://people.rit.edu/~gtfsbi/imm/Videos/II_4_1_Gene_Recombination-H264.mov

12. Additional diversity generated during ligation of gene fragments If ONLY the recombination events were used, number of different possible receptor/antibody specificities would be ~300 VJ x ~6000 VDJ = ~ 1.8 million Actual number of specificities far exceeds 1.8 million “Junctional “diversity adds an almost unlimited number of possibilities Happens during ligation of each set of gene fragments (V-J, D-J, V-DJ) Removal of 7-base heptamers (of the RSS) by RAGs End nucleotides of each DNA chain are joined to each other (“hairpin”) Hairpins are “opened up” by RAGs Result is two nucleotides from one of the two strands becoming part of the other strand This is a change in the reading frame (adds some more possible specificities) These four nucleotides are called palindromic (P) nucleotides The resulting base pair sequences would read the same from either end

13. Junctional diversity continued One or more P nucleotides can be excised by exonucleases Additional nucleotides (called “N” nucleotides) can be added by TdT (terminal deoxynucleotidyl transferase) Excision of of P nucleotides and addition of N nucleotides can (theoretically) generate 3X107 (30 million) additional possibilities during each gene fragment ligation

14. So, how many specificities? 300 VJ x 3x107 = ~ 9x109 possible light chain variable portions 6000 VDJ x 3x107 = ~18x109 possible heavy chain variable portions 9x109 x 18x109 = ~1.62x1020 possible antigen-binding sites Many are “unuseable” (do not generate sequences that can be transcribed) Still generates trillions of functional specificities Similar process for T-lymphocyte specificity

15. Antibody structure Using IgG as an example (other isotypes differ in the constant portions of the heavy chain) Two identical light chains (~220 amino acids in length) Two identical heavy chains (~440 amino acids in length) Carboxy-terminal and amino-terminal ends of each chain Interchain covalent, disulfide bonds H=H, H-L Molecule acts as a “unit”

16. End of Exam 2 MaterialStart of Exam 3 Material

17. Antibody structure Using IgG as an example (other isotypes differ in the constant portions of the heavy chain) Two identical light chains (~220 amino acids in length) Two identical heavy chains (~440 amino acids in length) Carboxy-terminal and amino-terminal ends of each chain Interchain covalent, disulfide bonds H=H, H-L Molecule acts as a “unit”

18. Antibody structure (continued) Hinge region (allows slight flexing) Exposes complement-activating site after binding to antigen Can adjust to different spacing of epitopes (antigens) Variable portions (VL, VH) VL+VH -> one antigen (epitope) binding site Constant portions Light chain – either kappa or lambda Heavy chain – M,G,A,E (rarely D, except for B-cell receptor)

19. Antibody Structure (continued) Antibody fragments (shows locations of functions) Papain cleavage Two identical Fab fragments (Fragment antigen-binding) One Fc fragment (Fragment crystalizable) Location of virtually all of the biological functions except binding to antigen

20. More “realistic” look at overall antibody shape “Antiparallel” folding of chains –> beta sheets Amino acids not in the beta sheet strands form loops Several non-covalent bonds between adjacent amino acids of the beta sheet chains One covalent (disulfide) bond between two strands ALL create very stable tertiary structure Beta sheets establish “domains” of the molecule Typical “immunoglobulin” properties Found in many other molcules Immunoglobulin “superfamily”

21. Antibody specificity at the molecular level Primary amino acid sequence of a variable portion Should see considerable differences between different specificities Compare sequences of different specificities Some parts of each sequence are more variable than other parts Named “hypervariable” (HV) amino acid positions also called CDR – complementary-determining regions Three HV sequences/variable portion (heavy and light chains) Less variable sequences called framework (FR) amino acids

22. Functions of HV and FR amino acids Compare primary and tertiary structure HV amino acids occupy three loops at “external” surface of variable portion Form the bonds with the antigen FR amino acids are the beta strands and other loops Provide overall structural stability of the variable portion

23. Specificity determined by amino acids in the HV positions and the positioning of these HV amino acids by the FR amino acids Variable portion shape and HV amino acids “unique” for each antigen Antigens to which BCR and antibodies bind are usually located at surfaces Each BCR and antibody specificity will bind to unique antigen Atoms of an antigen need not be adjacent to each other in the tertiary structure of the antigen

24. Overall shape of binding site is “complementary” to shape of antigen Maximizes the number of bonds that form between the antibody and the antigen (antibody “affinity)” All of these bonds are short-range, weak, non-covalent Electrostatic (ionic), hydrophobic, hydrogen, van der Walls

25. Even more diversity – somatic hypermutation During an antibody response to antigen memory B-cells will undergo somatic hypermutation under the influence of T-helper cells Random nucleotide point mutations in all three HV (CDR) regions of both light and heavy chains during transcription of the assembled variable portions Somatic hypermutations can accumulate over time Some result in a slightly improved affinity of the BCR and secreted antibody – this increase is another example of a “better” secondary immune response

26. Exams on front table – Exam average = 38.4/50Your exam 2 score is circled in RED on 2nd pageRed score on page 2 does NOT include any extra credit seminar points if earned(Exam 2 scored posted in MyCourses DOES include any extra credit seminar points)One ambiguous and two confusing questions (see link to exam for details) TWO Clicker questions at the start of class 1. The most important function of the hypervariable (HV) amino acids (sometimes called the CDRs -complimentary determining regions) in an IgG antibody molecule is which one of the following? 2. Treatment of one IgG antibody molecule with the enzyme, papain, produces which one of the following outcomes? Voting for your one favorite Athymic mouse finalist will take place after the clicker questions are completed

27. The most important function of the hypervariable (HV) amino acids (sometimes called the CDRs -complimentary determining regions) in an IgG antibody molecule is which one of the following? A. maintain the overall stability of the variable portion domains B. allow the IgG antibody to flex slightly, which can lead to complement activation and gives the antibody the ability to “adjust” the spread of its variable portions to accommodate slightly different spacings between identical antigens on the surface of a large, complex structure (e.g., on the surface of a bacterial cell C. gives the IgG antibody molecule several biological functions such as the ability to cross the human placenta, attach to Fc receptors on macrophages, and activate complement D. bind directly to the antigen for which that antibody is specific

28. Treatment of one IgG antibody molecule with the enzyme, papain, produces which one of the following outcomes? A. somatic hypermutation B. three fragments, two of which are identical (Fab) and one that can be crystallized out of solution (Fc) C. four separate polypeptide chains (two light and two heavy) that result from the hydration of the S-S disulfide bonds between these chains D. decrease in the affinity of the antigen binding sites

29. 2. If a macrophage has two different receptors on its surface, that macrophage will be able to directly attach to virtually every type of bacterial pathogen (except perhaps for those with a capsule). These two receptors are in addition to the repertoire of TLRs also expressed by these same phagocytes. These two additional receptors are which one of the following pairs? (select one letter as your answer) A. Fc receptor and C3b receptor B. LPS (lipopolysaccharides) receptor and “scavenger” receptor C. antigen-specific receptor and mannose receptor D. CXC8 and IL-12 receptor 3. If a macrophage has two different receptors on its surface, that macrophage will be able to indirectly attach to virtually every type of bacterial pathogen (even those with a capsule, using other molecules as a molecular “bridge” between the macrophage receptor and the pathogen). These two receptors are in addition to the repertoire of TLRs also expressed by these same phagocytes. These two additional receptors are which one of the following pairs? (select one letter as your answer) A. Fc receptor and C3b receptor B. LPS (lipopolysaccharides) receptor and “scavenger” receptor C. antigen-specific receptor and mannose receptor D. CXC8 and IL-12 receptor

30. 20. Successful neutralization of a virus could be accomplished by which of the following antibody isotypes? (select all answers that are correct)   A. IgM B. IgG C. IgA (the monomeric form found in the serum) D. secretory IgA Write +3 on the first page of your Exam 2, sign your name next to that +3 and turn the exam in when you leave class this morning. 3 points will be added to your Exam 2 score (in addition to any seminar extra credit points you may have also earned)

31. Our Four Finalists!!

32. In your face

33. How does my camouflage work (can’t see me, huh)

34. Yes, I am soooooo cute

35. Let me outta here!!!

36. A. In your face B. How does my camouflage work? (can’t see me, huh) C. Yes, I am soooooo cute D. Let me outta here!!

37. Importance of the Constant portions Light chain kappa and lambda – no definitive functions Heavy chains (some properties have been previously discussed) M is always used first unless isotype switching occurs Process for isotype switching is similar to somatic recombination except complete genes are selected and no nucleotide “events” during ligation

38. Importance of the Constant portions Light chain kappa and lambda – no definitive functions Heavy chains (some properties have been previously discussed) The B-cell receptor (uses only M or D)

39. Receptor or secreted antibody – which one? Determined by inclusion or exclusion of MC (membrane coding) sequence MC only associated with M or D (the only two isotypes used for receptors)

40. Polymeric forms of secreted antibody IgM – pentamer (with a “J” chain) 10 identical epitope binding sites/IgM antibody Particularly useful in “aggregating” many bacterial cells (more easily removed from the body)

41. Polymeric forms of secreted antibody Secretory IgA (sIgA) – dimer with a “J” chain Predominates in fluids associated with membranes (monomeric form in the blood) Moved across epithelium (acquires a “secretory” component) Secretory component thought to also contribute to resistance from degradative enzymes

42. Some important physical properties of the isotypes

43. Some important functional properties of the isotypes

44. NK cells and antibody-dependent cellular cytotoxicity (ADCC) NK cells can “kill” (usually via apoptosis) virus-infected cells and some tumor cells (see earlier discussion of inhibition-activation) NK cells also have receptors for Fc component of IgG antibodies IgG antibodies bind to antigens on the “target” cell NK cells bind to Fc of IgG -> sends “activation” signal to NK cell Result is killing of target cell

45. Some important functional properties of the isotypes

46. IgG from blood into tissues Does not require any vasodilation or other changes in the endothelial cell layer Involves special form of an Fc receptor (FcRn) that binds only IgG

47. Some important functional properties of the isotypes

48. IgG across the placenta NOT related to just the size of the molecule The amino acid composition of the constant portion is crucial sIgA predominates in breast milk

49. Antibodies from beginning to end “Naïve” B-lymphocytes establish receptor specificity (somatic recombination and junctional diversity) BCR binds directly to “accessible” antigen (usually surface of pathogen or large soluble molecule) B-cell internalizes, degrades, present peptides to T-helper cell (using MHC Class II) T-cell “help” (cytokines) initiate B-lymphocyte replication and differentiation into plasma cells Plasma cells synthesize, assemble and secrete antibodies (with specificity same as the antigen receptor on the original B-lymphocyte)

50. TWO Clicker questions at the start of class Which one of the following immunoglobulin isotypes is secreted as a pentameric antibody, has a “J” chain, is quite effective in activating complement, and can bind up to 10 different pathogens (e.g., bacteria)/one antibody molecule? Antibody-dependent cellular cytotoxicity (ADCC) can be a rather effective way to destroy virus-infected cells. ADCC involves Natural Killer cells and antibodies specific for non-self antigens on the surface of the infected cell. However, for the antibodies to be effective, they need to be which one of the following isotypes?

51. Which one of the following immunoglobulin isotypes is secreted as a pentameric antibody, has a “J” chain, is quite effective in activating complement, and can bind up to 10 different pathogens/one antibody molecule? A. secretory IgA (sIgA) B. IgG (but only two of the subgroups of this isotype) C. IgM D. IgE (especially if the pathogen is a parasitic worm)

52. Antibody-dependent cellular cytotoxicity (ADCC) can be a rather effective way to destroy virus-infected cells. ADCC involves Natural Killer cells and antibodies specific for non-self antigens on the surface of the infected cell. However, for the antibodies to be effective, they need to be which one of the following isotypes? A. secretory IgA (sIgA) B. IgG C. IgM D. IgE (but only if the infected cell is a mast cell)

53. Exploiting our knowledge of antibodies and antibody synthesis B-cell leukemia (uncontrolled replication of one B-cell) Generates a very high rate of one somatic recombination

54. Monoclonal Antibodies Tissue culture application to generate extremely high quantities of antibody of only one specificity (impossible to do in a living animal)

55. Some clinically relevant monoclonal antibodies Antibodies specific for a selected cell surface molecule e.g., CD4 (T-helper), CD8 (T-cytotoxic), CD25 (T-regulatory cell), tumor-specific antigen Isolate desired cells using fluorescence-activated cell sorter (FACS)

56. Remove tumor cells from bone marrow cell suspension Attach a magnetic bead to Fc portion of monoclonal antibody specific for non-self antigen on surface of tumor cells Mix cell suspension with these “magnetic” antibodies Antibodies will bind to only tumor cells (antibody specific for only antigens on the tumor cell) Pass entire cell suspension past a magnet Tumor cells are drawn to magnet Non-tumor cells keep flowing past

57. Modified monoclonal antibodies Genetically engineer a B-cell to generate a desired variable and/or constant portion that is more likely to be effective in or compatable with the human body (most monoclonal antibodies are made in mice) Chimeric – mouse variable + human constant Humanized – mouse CDRs + human FR and constant True human Mabs – very difficult to produce

58. Modified monoclonal antibodies “Chimeric” antibodies Variable portions of mouse antibodies Constant portions of human antibodies Rituximab – specific for a cell surface molecule (CD20) found only on human B-lymphocytes Used for certain B-cell lymphomas – tumor of B-cell IgG Mab specific for CD20 binds to all B-cells NK cells bind to human Fc portion of those IgG Mab B-cells are killed (includes tumor and healthy cells) - ADCC

59. Modified monoclonal antibodies “Chimeric” antibodies Variable portions of mouse antibodies Constant portions of human antibodies Rituximab – specific for a cell surface molecule (CD20) found only on human B-lymphocytes Used for certain B-cell lymphomas – tumor of B-cell IgG Mab specific for CD20 binds to all B-cells NK cells bind to human Fc portion of those IgG Mab B-cells are killed (includes tumor and healthy B-cells) - ADCC Plasma cells (lack CD20) continue to produce antibodies (so ongoing antibody production is not affected) Discontinue use of rituximab when all lymphoma cells are killed Published article - Mechanisms of killing by anti-CD20 monoclonal antibodies http://linkinghub.elsevier.com/retrieve/pii/S0161589007002623

60. Modified monoclonal antibodies Humanized antibodies Mouse CDR Human FR and constant As close to human as possible without the need to inject antigen into a human to generate antibodies Omalizumab – specific for Fc portion of human IgE antibodies Binds to Fc of IgE – prevents IgE from binding to mast cells (thus preventing mast cell sensitization and eventual degranulation) Pubished article - Omalizumab: A Monoclonal Anti-IgE Antibody http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1681435/

61. Modified monoclonal antibodies Human monoclonal antibodies Adalimumab (one of only a very few ever produced) specific for TNF-? (tumor necrosis factor-alpha) Binds to TNF-? and prevents further harm from excess TNF-? Often used for rheumatoid arthritis chronic joint inflammation caused by excess TNF-? Abbott Lab details about Humira (Adalimumab) http://www.humira.com/ More detail about Humira from MedicineNet.com http://www.medicinenet.com/adalimumab/article.htm