Term
| What is a protein domain? |
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Definition
| Amino acid sequence in a peptide that defines a region that regularly folds the same way and performs a predictable function, such as the catalytic site of an enzyme. |
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Term
| What is an immunoglobulin domain? |
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Definition
| Amino acid sequence in an antibody peptide that always folds into the same functional structure. |
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Term
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Definition
| A protein that is made by cells of the immune system. Some cytokines can boost the immune response and others can suppress it. |
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Term
| In figure 1, what are the immunoglobulin domains? |
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Definition
| Heavy chain and light chain variable and constant. |
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Term
| What is the difference between the variable domains and the constant domains? |
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Definition
| Constant domains are coded by the inherited genome, variable domains coded by recombined DNA. |
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Term
| Which domains bind to antigen and why? |
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Definition
| The heavy and light chain variable domains bind antigen because their sequence was selected out of the B-cell repertoire. |
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Term
| What does the structure of the constant domains tell you? |
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Definition
| What effector cell it will attach to. |
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Term
| What function does the Fc region of an antibody have? |
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Definition
| Fc fragment mediates effector activity. |
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Term
| What is an effector molecule of the immune system? |
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Definition
| Effector molecules bind to Fc and cause some effector processes to happen, such as complement-mediated lysis, phagocytosis, and cell-mediated cytotoxicity. |
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Term
| What are the 3 principle modes of action of therapeutic antibodies? |
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Definition
| 1. Blocking ligand receptor interactions. 2. Targeting cells for destruction through natural processes by the immune system or through radionuclides or toxins attached to the antibody. 3. Acting as signaling agonists- through apoptotic pathways. |
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Term
| What are the 2 ways that blocking antibodies can interrupt cytokine signaling? |
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Definition
| 1. Block by binding ligand so it can't bind to the receptor. Block by binding receptor (can just be there or activate specific pathways- act as ligands) |
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Term
| What sorts of signaling effects can antibodies have? |
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Definition
| Crosslinking receptors (depends on receptor system) to activate cell division or other responses, including apoptosis. Acts as a ligand that "dimerizes" receptors (antibody has two binding sites). |
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Term
| What is complement-dependent cytotoxicity? |
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Definition
| After antibody binding via Fab sites, complement factors attach to Fc (has to be intact and functioning) and form the membrane attach complex to form a hole in the target cell--> cell lysis. |
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Term
| Which antibody classes are involved in CDC? |
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Definition
| IgG (we just use these recombinant forms for therapy) and IgM. We want the IgG Fc effector function. |
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Term
| What effector molecules are involved in CDC? |
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Definition
| Complement factors (any molecule that can bind to an Fc receptor) |
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Term
| What 3 problems are seen when mouse monoclonal antibodies are used as therapeutic or diagnostic agents in humans? |
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Definition
| 1. Short half-life- recognized as a foreign protein. 2. Failure to trigger effector function. Mouse Fc domain is different enough from human Fc that wanted response may not occur. 3. Human anti-mouse antibodies- Hama syndrome. |
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Term
| What approach was taken to fix these problems when mouse monoclonal antibodies are used as therapeutic or diagnostic agents in humans? |
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Definition
| Genetic engineering approach to make chimeric antibodies that are partially human sequence and less immunogenic. |
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Term
| What is a chimeric antibody? |
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Definition
| A genetically engineered antibody with human constant domains (Fc 70%) and mouse variable domains (Fab 30%) |
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Term
| What advantages do chimeric antibodies have? |
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Definition
| Less immunogenic- still immunogenic enough to cause response though. |
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Term
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Definition
| Next step above chimeric- an attempt to minimize the mouse sequence by only cloning the mouse CDR sequence into a human variable domain. CDR loops are sufficient enough to bind antigen and have little immunogenicity associated with the final antibody (STILL CAN PROVOKE RESPONSE!!) |
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Term
| What 3 developments have led to the possibility of making fully human antibodies by genetic engineering? |
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Definition
| 1. Discovery of human variable region genes. 2. Successful expression of human variable region genes in E. coli. 3. Invention of phage display method. Able to generate Fab region found while screening for antigen affinity using human DNA. In theory should not have any immunogenicity because it's completely human, but STILL PROBLEMS if expression system is anything other than animal. |
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Term
| If you were determined to use mice to make fully human monoclonal antibodies, what would you have to do to the mice? |
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Definition
| Exchange natural mouse immunoglobulin genes for the human genes. Makes human IgG antibodies (All domains are Human DNA). |
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Term
| What is serum therapy and when was it used? |
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Definition
| The IV use of serum from a patient who is assumed to have immunity to the infectious disease of the patient. Very dangerous because person could react to therapy but it was mostly last resort anyway (person would die if didn't receive it). Primarily done before the age of antibiotics. |
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Term
| If serum therapy is still used, what is it used for? |
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Definition
| Today serum therapy is only used for emergencies such as acute lethal toxin (snake/spider venom) or virus exposure such as rabies or botulism. |
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Term
| What is post-exposure prophylaxis? |
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Definition
| Prevention of infection after known exposure to the agent. Ex. rabies. |
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Term
| What are the 5 Immunoglobulin classes? |
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Definition
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Term
| Which immunoglobulin class is the first produced in a primary immune system? |
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Definition
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Term
| Which immunoglobulin class is involved in histamine release? |
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Definition
| IgE- binds to basophils and mast cells, also principle Ig component in response to parasites. |
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Term
| Which immunoglobulin class is most abundant in serum? |
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Definition
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Term
| Which immunoglobulin class protects the fetus? |
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Definition
| IgG- has to do with neonatal Fc receptor, can be shuttled back and forth across the placenta. |
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Term
| What are the two main techniques used to make fully human, monoclonal antibodies today? |
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Definition
| 1. Transgenic mice (Makes human IgG antibodies in response to antigens injected into the mouse). 2. Phage display was the first to be adopted by pharm industry. Can use this to discover Fab regions for things that can't be injected into the mouse. Requires much more genetic engineering than mouse. Products of phage display are produced in chinese hamster ovary cells. |
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Term
| What is the most effective way of making a drug using monoclonal antibodies to treat viral infections? |
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Definition
| Making an artificial polyclonal mixture of monoclonal antibodies. Viruses rapidly evolve and change coat proteins to evade the immune system. Using a single Mab may be less effective if the virus is able to mutate. If you have a mix of monoclonal antibodies that have subtle differences then it would take longer for the virus to develop mutations to all of them and be eradicated before this could take place. |
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Term
| Which cytokine is associated with rheumatoid arthritis and how? |
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Definition
| TNF-alpha accumulates in joints where it causes inflammation. Tends to accumulate in high levels in joints and stimulate inflammatory cells to attack joint tissues. |
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Term
| In table 1, what are the 2 most frequent general disease indications for approved monoclonal antibodies? |
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Definition
| Cancer and Immunomodulatory drugs are the two most frequent indications for monoclonal antibodies. |
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Term
| What is the general purpose of using therapeutic antibodies to treat cancer? |
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Definition
| Direct and indirect destruction. |
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Term
| What are the two main targets of the antibodies (to treat cancer)? |
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Definition
| The cell itself to kill by effector cells or toxic molecules attached. Or its blood supply- the tumor can only grow so large before the cells become hypoxic and stimulate blood vessel growth. |
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Term
| How can you use radioactive elements and antibodies together to kill cancer cells? |
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Definition
| Tightly bind a radionucleid atom to the antibody without blocking the Fab binding site. This delivers Isotope to target cell but CANNOT affect Fab. |
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Term
| What do you need in an antibody in order to kill tumor cells by natural immunoglobulin effector functions? |
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Definition
| Intact functional Fc region to activate ADCC, CDC, and Phagocytosis. |
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Term
| Why do antibodies that block VEGF signaling inhibit tumor growth? |
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Definition
| VEGF causes a blood supply to form that nourishes the tumor (blocking VEGF inhibits this process). Only significant for solid tumor forming cancer- not a lot of effect in leukemia. |
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Term
| What approved antibodies work by blocking VEGF? |
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Definition
| Avastin (bevacizumab) and Lucentis (ranibizumab) |
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Term
| What is a naked antibody? |
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Definition
| A plain (natural state) antibody molecule with nothing attached to it (glycosylation can be attached to it) |
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Term
| How does a naked antibody differ from other types of therapeutic antibodies? |
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Definition
| A naked antibody requires natural Fc-mediated effector function to have a therapeutic effect. |
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Term
| When naked antibodies kill cancer cells via effector cell cytotoxicity,how does the killing actually happen? |
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Definition
| Cytotoxic and phagocytic cells bind the Fc region and kill or engulf the cancer cell. |
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Term
| What are bispecific antibodies and how could they be used? |
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Definition
| Bispecific antibodies have 2 different antigen specificities (Fab regions). Antigen bound to heavy chain domain is then carried to the cell that will recognize and process it using the other (light chain domain) Fab domain |
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Term
| What effector cell population would be the best target for bispecific antibodies? |
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Definition
| A cytotoxic effector cell |
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Term
| What adverse side effect of naked antibody therapy is possible? |
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Definition
| Cytokine storm (cytokine release syndrome). Fc region overactivates lymphocytes and everything gets attacked (host) |
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Term
| What approach is taken to avoid cytokine storm? |
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Definition
| Fc receptor can be mutated to prevent activation of immune effector cells and cytokine secretion. |
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Term
| How can small antibodies (short chain Fv) lacking the Fc region be used in therapy? |
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Definition
| Neutralizing toxins or over produced substances. Blocking ligand receptor interactions. No possibility of Fc mediated response--> lowered S/E!! |
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Term
| Which antibody fragments can be used as blocking agents? |
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Definition
| Fv (engineered variable fragment), Fab (natural or engineered), Fab2 (natural or engineered- additional Fab instead of Fc portion). Don't need Fc portion to block!! |
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Term
| What two major advantages does the phage library approach have over the traditional monoclonal antibody approach (injecting mice and making hybridomas)? |
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Definition
| 1. More screening capacity can give better specificity and affinity. Phage particles that bind to antigen are harvested and then cloned with human DNA. Mouse you have to screen the lymphocytes to see what antibodies are produced. 2. Haptens, toxic drugs, and other toxic substances can be used as antigens. These would kill mouse and most drugs wont induce a response if not haptenized. Will get lots of antibodies to protein carrier instead of actual drug. |
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Term
| What approach in engineering antibodies is taken to increase avidity? |
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Definition
| Producing single chain Fv molecules ant then grouping them into dimers and trimmers. Possible to make even more multiples (more multivalent= more avidity) avidity= combined strength of multiple bond interactions |
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Term
| What effect on crosslinking ability does an increase in avidity have? |
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Definition
| Increasing avidity increases crosslinking activity. Avidity is the strength of binding one ligand to a receptor. Only makes sense when multiple receptors are present and the ligand can bind to these (power of stickiness) Different from affinity |
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Term
| Are complete antibody molecules able to penetrate tissues easily? |
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Definition
| NO- naked antibody molecules or delivery antibodies are TOO BIG to easily penetrate NORMAL cells/tissue. If inflammation or swelling, however, ALL plasma proteins can get in!! |
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Term
| What 2 key factors affect the therapeutic efficacy of antibodies? |
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Definition
| 1. Stability (half-life)- what killed the monoclonal mouse approach. 2. Immunogenicity- mouse recognized as foreign is and why they are cleared so fast and could lead to an over-reactive response. |
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Term
| What is a pharmacokinetic problem posed by small antibody fragments? |
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Definition
| Rapid clearance from the circulation by kidney filtration. They are so small that they easily pass through the glomerulus. |
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Term
| Why do complete (naked) antibodies tend to have a long serum half-life? |
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Definition
| Presence of the Fc region allows binding of neonatal Fc receptor that prevents destruction of antibody. Most monoclonal antibodies are IgG. IgG can pass through the placenta and protect the baby because they are shuttled in and out due to the neonatal Fc receptor (FcRn). Antibodies are constantly taken up and spat back out by this system. After being taken up they can be degraded by lysozomes or if in good condition return to the cell surface and recycle back- WHY the HALF-LIFE can be WEEKS. |
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Term
| What modification can be made to increase the serum half-life of antibodies and their fragments? |
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Definition
| Addition of polyethelene glycol polymers (PEG-ylation) |
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Term
| How does PEGylation work? |
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Definition
| It increases the molecular weight (size) above the kidney filtration threshold and lowers immunogenicity. |
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Term
| What 2 immune potentiation properties will the best vaccines for infectious disesase have? |
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Definition
| Stimulation of both (1)antibody production and (2)T-cell activation. The more powerful response is the activation of T-cells. This doesn't always happen and is why some vaccines are not as successful. |
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Term
| What dose a "troybody" do and how does it work? |
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Definition
| Antigenic protein is bound to Fc while Fab targets specific T cells. We are mimicking the function of an antigen presenting cell doing this. We are directly stimulating effector T-cells to deal with the antigen. |
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Term
| What other way (besides troybody) can you deliver antigen to immune effector cells? |
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Definition
| Attach the antigen to another part of the antibody molecule to allow Fc-mediated uptake. The antigen is not attached to either the Fab or Fc portion. Can direct which type of effector cell that is targeted through Fc component. |
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