Antibodies - Lecture notes ? PDF

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Antibodies “proteins of the adaptive immune response” -> globular proteins called immunoglobulins Basic structure: Y shapes molecule arms give specificity -> high affinity and specificity binding (binds to the right thing and binds well) Basic Function: binds foreign mole...

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Antibodies! “proteins of the adaptive immune response” ! -> globular proteins called immunoglobulins! Basic structure: Y shapes molecule! arms give specificity -> high affinity and specificity binding (binds to the right thing and binds well)! Basic Function: binds foreign molecules e.g. antigens! able to do this via: binding versatility, binding specificity, biological activity!

IgG is used as the prototype antibody to explain the basic structure of all antibodies.! 2 heavy chains (H, 50kDa) and 2 light chains (L, 23kDa) = antibody, linked by disulphide bridges and non covalent interactions.! Each chain (light and heavy) is divided into two regions, the variable (V) and constant (C) regions.The amino-terminal end contains the V region [NH2], while the carboxyl-terminal end contains the C region [COOH.] Thus the tips of each chain contains the variable region. ! Light chain: two domains, CL and VL (the constant and the variable regions.) Each antibody only contains one type of light chain, either:! a kappa (k) chain from chromosome 2 ! a lambda (λ) chain from chromosome 22! kappa:lambda type antibodies should be 2:1 in serum. Information about wether the light chain is k or λ is found in the constant region. ! The variable region = antigen binding site. Specifically, these sites are called CDRs, “hypervariable regions or complementarity-determining regions.”! • 3 CDRS per variable region! • about 10aa long! • rest of the variable region (85%) is made of framework residues, with restricted variability -> these define the positions of the CDRs! • CDRs are exposed on the surface of the antibody by folding! • CDRs form a cleft = antigen binding site! 1 variable region per chain, overall 4 chains (2 light, 2 heavy), 3 CDRs per variable region so 12 CDRs in an entire antibody!!

the CDRs define the specificity of the antibody ! Heavy chain = four domains, one variable (VH) and three constant (CH.) Variable domain has 3 CDRs and the rest is framework residues. The three C-region units show strong homology to each other and to the C region of the light chain. ! Within the CH2 domain there is glycosylation action that allows the two CH2 domains to associate together. ! There are five types of mammalian immunoglobulin heavy chain: γ, δ, α, μ and ε.*They define*classes*of immunoglobulins:*IgG,*IgD,*IgA,*IgM*and*IgE, respectively. The only difference between the five classes is in their heavy chain constant regions. The hinge region is located between the CH1 and CH2 domains. Only IgG, IgA, and IgD antibody molecules have hinge regions.! μ and ε have more amino acids and thus IgM and IgE have an additional CH4 domain. ! ! ! Fab fragments: peptide chains that are still able to bind antigens; the product of hydrolyses of the peptide bonds in an antibody by papain action. Contain the entire light chain and part of the heavy chain. ! fragment: [aka fragment crystallisable] Contain only the heavy chain after the hinge gion, have no antigen binding activity, constant region, ! ain action: cleaves an antibodies’ peptide bonds, giving two Fab fragments for every one Fc fragment. IgG was initially used in Porter’s 1959 experiment.! Pepsin action: hydrolyses different sites on IgG than papain, giving one large (100kDa) fragment and many small ones. F(ab)2 degrades into 2x Fab’ if the disulphide bridges are disrupted (reducing agents.) ! F(ab)2: name given to the large fragment yielded from pepsin and IgG, can still bind the antigen. Can be cleaved additionally to give two Fab-like fragments called Fab’. Each of the Fab’ fragments is made up of the entire light chain and a slightly longer part of the heavy chain.! Each domain in an antibody has a two beta pleat tertiary structure with a hydrophobic interior. Domains are linked by disulphide bridges (covalent linkage.) The CDRs are held out of the structure are loops which form the antigen binding site. !

VDJ recombination Three loci in the genome code for antibodies:! a kappa (k) chain from chromosome 2 ! a lambda (λ) chain from chromosome 22! a heavy chain from chromosome 14! the light chain therefore does not have a single gene and must be spliced together -> example of DNA splicing! For each light chain to be made we need, from the DNA:! a leader sequence for cell signalling (removed in post-translational modifications)! a variable sequence (V)! a J sequence! a C sequence! variable, diversity and joining gene segments are added together to give light and heavy chains -> provides diversity among immunoglobulin molecules and results in new amino acid sequences. ! Each heavy chain and light chain gene contains multiple copies of three different types of gene segments for the variable regions of the antibody proteins. For example, the human immunoglobulin heavy chain region contains 2 Constant (Cμ and Cδ) gene segments and 44 Variable (V) gene segments,[2]*plus 27 Diversity (D) gene segments and 6 Joining (J) gene segments.[3]*The light chains also possess 2 Constant (Cμ and Cδ) gene segments and numerous V and J gene segments, but do not have D gene segments.! in making the kappa light chain, V and L regions are paired together so if you take L2 you also take V2, and L30&V30 e.g. There are about 40L&V regions to pick from. A J sequence is chosen out of five possibilities. The C region is added. ! the heavy chain DNA has 65V regions, 27D regions and 8J regions -> any of them can be spliced together to make the mRNA for the heavy chain. Both C regions are added!! Unwanted DNA regions are lost through deletion to give the primary mRNA transcript. In heavy chain formation both constant regions are added!! THUS, one B cell produces one antibody -> this antibody has a distinct variable region due to VDJ recombination. The nature of the somatic recombination of the B cell DNA means that this B cell can only produce antibodies with the specific variable region pattern initially made. All other VDJ regions in the DNA other than the chosen three are removed.!

[While diploid cells have two copies of every immunoglobulin gene, only one of the two is expressed in a given B-cell or plasma cell for each of the light and heavy chain (the other allele is either rearranged aberrantly and cannot be expressed, or is not rearranged at all). This is an unusual and important feature of immunoglobulins (and the TCR); although recent work has shown that there exist a number of other genes that also exhibit this behavior.]! ! Class switching Changes antibody production from one class to another by changing the constant region in the heavy chains -> class switching does not affect antigen specificity as the variable regions do not change. The antibody-effect molecule interactions change.! IgM and IgD are made at first, using constant regions called μ and δ. Class switching allows IgA, IgG and IgE antibodies to be made. ! In class switching the unwanted μ or δ heavy chain constant region*exons are removed and substituted with a γ, α or ε constant region gene segment. ! Switching is unidirectional--an IgG-producing cell cannot go back to production of IgM, for instance. ! Why class switch?! -> over the course of an immune response, the affinity of antibodies to the antigen increases. Thus, it starts at a low affinity. (affinity maturation.) The only class of antibodies able to compensate for this lowered affinity is the IgM. However, IgM is polymeric and forms a pentamer and is costly to the cell to produce. The process of affinity maturation can allow the expensive IgM to be replaced with more economical IgG-like antibodies. ! There are five classes of antibodies based on the structure of their heavy-chain C domains. All antibody classes have either l or k light chains.! b. IgG is the major antibody in the blood, but it is able to enter tissue spaces and coat antigens, speeding antigen uptake.! c. IgA concentrates in body fluids to guard the entrances of the body.! d. IgM is the largest antibody; it tends to remain in the blood, where it can lead to efficient killing of bacteria.! e. IgD remains membrane-bound and somehow regulates the cell’s activation.! f. IgE is found in trace amounts in the blood, but it still triggers allergies.! CDR-H3 is particularly variable: V/D/J splice site! Effectors Fc receptors are found on cell surface membrane (e.g. macrophages) and bind to the Fc fragment on antibodies -> the heavy chain constant 2 and 3 (&4) domains. Binding of an antibody to the Fc receptor can trigger phagocytosis of the antibody and its bound antigen. ! FcgR (bind IgG): primarily on neutrophils, macrophages and monocytes. Phagocytose IgG coated pathogens.! FceR (bind IgE): primarily on eosinophils, basophils and mast cells. Trigger histamine! release (swelling lets macrophages into tight blood vessels) from intracellular granules (mast cells.)! FcaR (bind IgA): neutrophils, monocytes, macrophages and eosinophils. Phagocytosis, antibodydependent cell-mediated cytotoxicity (ADCC), release of superoxide and inflammatory! mediators!

Complement system: protein cascade in the blood results in phagocyte stimulation. It is initiated by IgG and IgM binding to antigens. C1q is part of the protein complex that binds the antigenantibody complex. The complement system is now initiated. ! C1q must bind at least two Fc regions to activate -> as IgM is a pentamer is it more likely to trigger the cascade. ! ! IgM and IgA: polymeric structure ! IgM = pentamer via a J chain (not to do with genetics) ! IgA = dimer via a J chain !

Immunoglobulin fold Beta sandwich creates a 110aa domain where the two beta sheets have a 30 degree incline -> very stable and preserved over many structures aka “superfold.” It has a central hydrophobic core and a disulphide bridge between the two sheets. ! Each of the six domains in an antibody adopts the immunoglobulin fold: VH, CH1, CH2, CH3, VL, CL.! The fold is present in the variable region where it supports the three CDR loops at one end of the fold. At the other end of the structure is the amino and carboxyl termini, which continue the light and heavy chains. ! Barrel Variable regions in the light and heavy chains interact via 5 stranded sheets to form a barrel. ! Chothia’s analysis: examined eight structures and found that:! the CDRs, despite being hypervariable in sequence are surprisingly conserved in structure for some of the CDRs (e.g. CDRL1) even though the length varies considerably, the overall! structure is maintained with variation in length being accommodated in a localised area of! the loop.! the conformation is defined by the presence of certain key residues within the loop and! packing against the loop = framework residues. Providing these are conserved, the rest of the sequence of the loop can vary widely while maintaining the same conformation.! framework residues allow CDRs to have a conserved overall structure despite their hyper variable sequence.! key residues are more important than CDR length, as difference in length is handled by extending the length of the hyper variable loop!

NOTES ON HUMANISATION, CHIMERAS and what was in the rest of lecture 2! !

How was the structure found? Monoclonal and polyclonal antibodies do not crystallise -> we need purity and all antibodies have variable domains! ! Polyclonal – mixed antibodies binding same antigen! Monoclonals – identical antibodies! Understand how the characterisation of antibody structure led to the comprehension of antibody function.! 2. Distinguish between the overall structure and the fine structure of antibodies.! 3. Describe the variable and constant regions of an antibody’s light and heavy chains.! 4. Explain the organization of the variable regions of an antibody’s light and heavy chains; define hypervariable regions and domains.! 5. Name and compare the biological and chemical characteristics of the five classes of antibodies.! 6. Discuss the differences in the biological effector functions of antibodies.! 7. Contrast conventional antibody and monoclonal antibody development; conceptualize the procedure for monoclonal antibody screening; and discuss hybrid monoclonal antibodies.! [Antibody-containing serum is called antiserum, in contrast to normal serum (the clear yellowish fluid collected when whole blood is separated into its solid and liquid parts) that does not contain antibody to a specific antigen.]! Assembly of the heavy and light chains into a typical IgG-like subunit (H2L2) by formation of disulfide bonds is a spontaneous process - no specific enzymes are thought to be required, although a member of the HSP70 class of molecular "chaperonins" (the Heavy Chain Binding Protein, or BiP) is known to facilitate the proper folding of the heavy chains prior to their association with light chains. ! Assembled H2L2 molecules move through the RER to the Golgi apparatus, and into the postGolgi vesicles. These vesicles then fuse with the external cell membrane and release their contents, resulting in secretion of Ig from the plasma cell. 5) Carbohydrate is added to H-chains in a well-defined order, beginning on nascent chains while they are still attached to ribosomes, and continuing throughout the process of movement through the cell right up to the point of secretion. Carbohydrate is added to asparagine residues which are part of a specific recognition sequence, namely an asparagine separated by one amino acid residue from a serine or threonine!

47. Describe the ‘immunoglobulin fold’. Superfold often found in proteins, consists of a beta sandwich two beta sheets are intertwined at 30 degree incline to each other with a hydrophobic pocket! a disulphide bridge links the beta sheets! the amino and carboxyl termini are on one end, joined to the rest of the light and heavy chains, whilst the other end supports the 3 complementarity determining regions in looping out of the antibody to form the antigen binding site. !

48. In the context of antibodies, what are ‘complementarity determining regions’ and what is their function? part of the variable chain in antibodies (present in both the heavy and light chain)! three CDRs are found per variable region (the rest of the region is framework residues)! a single antibody thus has 12 CDRs! once the territory structure is achieved CDRs form loops that face outward of the structure ! The loops are stabilised by the immunoglobulin fold! they are the antigen binding sites on the antibody, giving it specificity! they are also called hyper variable regions because they have very diverse amino acid sequences their diversity arises from VDJ recombination, a mechanism of DNA splicing that puts together new variable, diversity (only in the heavy chains) and joining gene segments to create the light and heavy chains. !

49. Provide three examples of the application of antibodies. Western blotting: make or find antibodies specific to the protein of interest and use these antibodies to select the protein from gel membrane. Another antibody selects for the first and produces a colour change -> labelling and detection! antigen finding: used in clinic to detect the antigen, high specificity allows low concentration to be used. ! radio-labelling: radio label Fab fragments which can be tracked throughout the body, will still bind to it’s antigenic target without raising an immune response e.g. neuroblastoma.! & injected before surgery to held guide doctors towards the tumour using a handheld gamma probe e.g. anti-CEA antibodies used in colorectal cancer. ! cancer drug: Trastuzumab binds to EGF receptor, which is over expressed in breast cancer! reopro: binds platelet fibrinogen receptors. Prevents clumping and reclogging of coronary! arteries after angioplasty. ! 50. What are abzymes? antibodies with a catalytic effect ->! would make great drugs as they can target molecules but have had little commercial success! able to select between optical isomers, which normal chemical drugs cannot accomplish. ! 51. What are FAB fragments and how are they produced from whole antibodies? region on the antibody that bind antigens ! consists of one variable region and one constant region on both the light and heavy chains, thus consist of an entire light chain and part of a heavy chain! fragmented through action of the enzyme papain, which hydrolyses peptide bonds in the hinge region to digest the antibody into three pieces: 2x Fab and 1x Fc fragments. !

52. Why do Fc fragments of polyclonal antibodies crystallise? polyclonal antibodies have different variable regions but are from the same class and thus have identical constant regions. Since Fc fragments consist of the constant domains of the heavy chain (domain 2 and 3 (&4)) they will all be identical even in a polyclonal group. This uniformity allows the Fc fragments to crystallise. ! 53. What are ‘chimeric human antibodies’ and why are they produced? Antibodies where the variable regions, the antigen binding domain, is taken from one species whilst the constant regions are taken from another. Usually the constant region is human whilst the CDR loops and the rest of the variable region is from another species. ! They are a cheaper alternative to humanisation and often used in early stage research. They also allow the use of antibodies in animal models e.g. a rat antibody can be made to target a mouse receptor. ! 54. Describe the process of antibody humanisation. use recombinant DNA to alter the amino acid sequence of monoclonal antibodies from non human species to attenuate them to the human immune system.! via intermediate: e.g. mouse Fab spliced to human Fc fragment: the chimera antibody can be humanised by altering the sequence of the Fab region. As the CDR regions confer specificity to the desired antigen their sequence cannot be changed but the framework residues around the 3 CDR regions can be altered. Parts of the Fab framework that differ between mouse and human and altered to make them more human through mutagenesis. ! 55. What did Kabat and Wu find out about antibody sequences? assembled amino acid sequence information about light chains into a variability plot showed 3 regions of hyper variability ! suggested that these regions were the complementarity determining regions of the antibody, and acted as the antigen binding site! 56. What did Chothia find out about the structure of CDRs? the CDRs, despite being hypervariable in sequence are surprisingly conserved in structure for some of the CDRs (e.g. CDRL1) even though the length varies considerably, the overall! structure is maintained with variation in length being accommodated in a localised area of! the loop.! the conformation is defined by the presence of certain key residues within the loop and! packing against the loop = framework residues. Providing these are conserved, the rest of the sequence of the loop can vary widely while maintaining the same conformation.! framework residues allow CDRs to have a conserved overall structure despite their hyper variable sequence.! difference in length of CDRs are handled by extending the length of the hyper variable loop!

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