Function and Importance of Antibodies Part 2 Flashcards
B cell receptor (BCR)
The receptor provides signals that activate the B cell, it starts secreting pentameric IgM. The antigen is then taken up by receptor mediated endocytosis, as B cells are not phagocytes
Antigen presenting cells (APCs)
Cells that bridge the gap between innate and acquired immunity. They present peptide fragments of antigen on their cell surface to T cells. All cells are capable of presenting the antigens of viruses and intracellular bacteria
Professional antigen presenting cells
Present antigens of extracellular and phagocytosed pathogens. Includes dendritic cells, macrophages, and B cells
TH2 cell activation
TH2 Helper T cells activate B cells and lead to antibody mediated immunity during the adaptive immune response. Professional APCs can present antigens to T cell receptors via MHC molecules. When the antigen binds the TCR, APCs secrete cytokines like IL-4. IL-4 activates the naive T cell into a TH2 cell. T cells can then go on to activate B cells. Endocytosis of an antigen send signals into the B cell that cause it to start secreting pentameric IgM, which is secreted in the early immune response. At this point, the B cell is not activated and has not undergone somatic hypermutation
Effector B cells (2)
Once the B cells are activated
1. Plasma cells
2. Memory B cells
Plasma cells
B cells that have undergone somatic hypermutation and are highly specialized for making large quantities of antibody with one specificity. The B cells that produce antibodies of higher affinity have been selectively expanded. Each plasma cell individually produces antibodies that are monoclonal. A group of plasma cells that each secrete antibodies of a different specificity constitute a polyclonal response. Antibodies also undergo a class switch, typically to IgG
Plasma cell structure
In plasma cells, almost the entirety of the cytoplasm is filled with endoplasmic reticulum. This is so the plasma cells can make large quantities of antibodies (proteins)
Memory B cells
Long lived B cells that activate rapidly upon a secondary encounter with a pathogen. They can directly activate T cells via antigen presentation, with no need for the dendritic cell middleman or activation by helper T cells. They generate a much more rapid response to antigens and generate immunological memory
Immunological memory- first exposure to antigen
- When we are first exposed to a pathogen’s antigens, it stimulates the immune response
- Causes the production of plasma cells to fight off infection
- Memory B cells are also produced
Immunological memory- second exposure to antigen
If you are ever infected with the same pathogen again, memory B cells are rapidly activated. This stimulates the production of an even larger pool of plasma cells and memory B cells. This response can occur so quickly that you don’t even know you’ve had an infection
Antibody classes produced in the immune response
IgM spikes first, then IgG- the IgG response is longer lasting. IgM is produced before somatic hypermutation and class switching have occurred. When we have already been exposed to a pathogen and have memory B cells, we have a much smaller peak of the IgM response. The IgG response occurs much more quickly and is larger.
How do vaccines work?
They stimulate a primary immune response and the creation of memory B cells to protect against a pathogen. Sometimes boosters are given in order to increase the pool of memory B cells. When the pathogen tries to infect, the memory B cells are activated rapidly
Types of vaccines (4)
- Killed whole organism- have all of the pathogen’s antigens
- Live attenuated whole organism- usually viruses. The viruses are made non-pathogenic for the vaccine
- Subunit vaccine
- mRNA vaccine
Subunit vaccine
Based on a single protein antigen from an organism. The vaccine contains one major antigen of a pathogen
Recombinant OspA
Only expressed in ticks- outer surface protein A (OspA) is the major antigen in the bacteria causing Lyme disease. It predominates on B. burgdorferi surface in culture. A recombinant form is used for the Lyme disease vaccine. One problem with this is that when the infectious agent is transmitted from ticks to humans, OspA ceases to be expressed.
Lyme disease vaccine
A subunit vaccine that is remarkably effective- 72% and 92% effective with and without adjuvant in clinical trials. Afforded protection via antibody-pathogen interactions in the tick vector. It activates memory B cells that produce antibodies against OspA and attack the Borrelia while it is still in the tick vector (while the tick is feeding). Second generation OspA vaccine that eliminates cross-reactive N-terminus
Tick transmission of B. burgdorferi
In non-feeding ticks, Borrelia may be present in the midgut, in very low numbers. At this point, Borrelia is predominantly expressing OspA/OspB on its surface. As a tick begins feeding on mammalian blood, the mammalian blood enters the midgut. This dramatically changes the environment for the spirochetes in the midgut- the temperature increases to mammal body temperature and the pH becomes slightly more acidic (7.5 to 7). There are also many blood components that are entering and changing the environment. All of these changes during feeding cause Borrelia to cease its expression of OspA/OspB and begin expressing OspC on its surface. At this point, Borrelia enters the salivary glands of the tick, then enters the mammal
How does the Lyme disease vaccine work?
People who have received the vaccine have memory B cells that secrete anti-OspA antibodies in their blood. When their blood floods the midgut of a tick, the antibodies will bind to an kill OspA expressing Borrelia, before they even have the opportunity to start producing OspC instead. Since tick transmission can occur over a period of hours, this illustrates the speed of the antibody response from memory B cells
Relapsing fever
Another type of Borrelia infection- these Borrelia are known to undergo antigenic variation. The spirochetes circulate in the blood, with one antigen being predominantly expressed, but others are present at lower numbers. If you have an immune response to the dominant antigen, the other antigens are still left over. This antigenic variation causes the waves of infection and fever, since there must be multiple immune responses to the different antigens. Therefore, when creating a subunit vaccine, you want to make sure the antigen is invariant and non subject to antigenic variation
COVID-19 vaccine
Pfizer and Moderna- mRNA based vaccines. The mRNA is delivered in a lipid nanoparticle, and host cells translate the mRNA to spike protein. When the spike protein is presented with MHC, it leads to B cell activation and antibody production
How does the COVID-19 vaccine work? (6)
- The vaccine injects COVID mRNA, which is contained in a lipid nanoparticle. Delivered to cells
- Once in the cells, the mRNA is translated to the COVID spike protein and presented on MHC
- A naive T cell recognizes the antigen. The target cell secretes IL-4, activating the T cell into a TH2 cell
- B cells also take up the mRNA and produce the spike protein, which is recognized by TH2 cells.
- TH2 secretes IL-4 and IL-5, activating the B cell
- Plasma cells, anti-COVID spike proteins, and memory B cells are produced
Lipid nanoparticles
Lipid nanoparticles are mostly cationic phospholipids. Some are
Polyethylene glycol (PEG) modified lipids or modified cholesterol (DP-cholesterol). The nanoparticles are designed to be easily taken up into cells
Passive vaccination
The administration of antibodies during an infection. This is different from active vaccination, as it’s therapeutic instead of prophylactic. The antibodies come from another source and are meant to treat an infection that has already occurred. Without active vaccination, infection does not happen or is subclinical
Polyclonal antibodies
A variety of different antibodies, each of a different specificity. Our natural immune responses are always polyclonal
Monoclonal antibodies
Antibodies of 1 specificity, which recognize 1 and only 1 epitope. Individual plasma cells produce monoclonal antibodies during natural immune responses
How are polyclonal antibodies generated?
When developed for medicine or research, polyclonal antibodies have antigen binding sites that are against 1 antigen with multiple epitopes. The epitope targets are usually unknown, and the affinity is unknown. The affinity is generally considered to be of lower affinity than monoclonal antibodies. The goal is high titer, high affinity antisera
Process of polyclonal antibody generation
An organism (mice, rats) is injected with an antigen and produces antibodies as part of the immune response. The animal’s blood is taken, which contains the polyclonal antibodies
How are monoclonal antibodies generated?
Monoclonal antibodies are always against 1 epitope. When developed for medicine or research, this means that the epitope target has been characterized and the affinity has been measured. Monoclonal antibodies are more of a known quantity than polyclonal antibodies
Process of monoclonal antibody generation
Myeloma cells are fused with primary spleen cells (a source of B cells). A selective medium for fused cells is used. Myeloma cells lack the ability to synthesize hypoxanthine-guanine-phosphoribosyl transferase (HGPRT), an enzyme necessary for salvage synthesis of nucleic acids. Aminopterin also inhibits their de novo purine synthesis pathway. Therefore, the myeloma cells cannot synthesize nucleic acids, and will not grow unless fused w/ B cells.
Why can’t primary B cells be used to generate monoclonal antibodies?
These cells do not have the ability to grow due to the Hayflick limit
Selective medium used in the generation of monoclonal antibodies
Uses HAT medium- hypoxanthine, aminopterin, thymidine
How mAbs are generated
Spleen cells cannot grow in vitro, while myeloma cells cannot synthesize nucleic acids and cannot grow in HAT medium. Therefore, we try to fuse the cells together. With a successful fusion, the cells will grow in the HAT medium.
Hybridoma cells
A hybrid of the fused spleen cells and myeloma cells. These cells have the qualities of cancer cells (they can grow forever) and B cells (they can produce antibodies)
Electrofusion
When 2 cells are brought into contact by dielectrophoresis (high frequency AC). When cells are brought together, a pulse voltage is applied. The pulse voltage causes membrane permeateation, the membranes combine, and the cells fuse. The nuclei fuse later on
Polyethylene glycol (PEG) fusion
The simplest but most toxic method of monoclonal antibody generation. PEG induces agglutination and cell to cell contact, and fuses the cell membrane and intracellular membranes. Can lead to the uncontrollable fusion of multiple cells
Why are monoclonal antibodies favorable candidates for drugs? (2)
- mAbs have high specificity & affinity- have little-to-no pleiotropic effects
- mAbs can be produced at high levels
Different parts of Abs & their functions (4)
- Whole antibody
- Fc fragment- effector functions
- Fab- antigen binding site
- Single chain variable fragment (scFv)- variable heavy and variable light chains joined by a flexible linker
Chimeric monoclonal antibodies
When mouse variable regions are fused to human constant regions. While this is better than a mouse monoclonal antibody, they are still 25% mouse in origin. There is a high success rate of monoclonal antibodies binding well to the target, though nothing about the variable region has changed. Produced via recombinant DNA
Humanized monoclonal antibodies
Mouse CDRs with human framework regions & constant regions, so these antibodies are greater than 90% human. Produced via recombinant DNA. Have low immunogenicity since the antibodies are less foreign- CDRs are the most variable region of an Ab anyway
Drawbacks of humanized monoclonal antibodies
These antibodies can result in a loss of efficacy and specificity. They use framework regions that have high homology to the mouse. This preserves structure of the antigen binding sites, but may involve a lot of trial and error. Better than mouse, better than chimeric, but still immunogenic because the antibody is not 100% human
Transgenic mice
Mice that have been genetically altered to produce fully human monoclonal antibodies. Regeneron produces a lot of their biologics this way
Phage display
Random screening of human variable regions- Humira was developed this way
Antibody phage display
Start with a group of healthy volunteers, take their blood, and isolate antibodies from the blood. You can specifically isolate the heavy and light chain variable regions. These regions undergo PCR to amplify them, and they then undergo cloning so they will be expressed on the surface of bacteriophages. We create a “phage library” where each phage expresses different human variable regions
Antibody phage display- purification
After the human variable are expressed on bacteriophages, they undergo a purification process. The phages are incubated with a surface or bead that is coated with the antigen we want to make the antibody against. The phages with the variable region complementary to the antigen will bind as they are run over the surface. We elute the bound phages and amplify the variable regions in E. coli. The selection process is repeated 2-3 times to ensure purity
mAb-based drugs terminology (4)
- “-ximab” is a chimeric antibody
- “-zumab” is a humanized antibody
- “-xizumab” is a humanized antibody
- “-umab” is a fully human antibody
Reopro (Abciximab)
Chimeric antibodies that bind the glycoprotein 2b/3a receptor. Inhibits platelet aggregation and is important after coronary artery procedures. Inhibits blood clotting
Avastin (Bevacizumab)
Humanized antibody that binds VEGF-A. Prevents angiogenesis, which is therapeutic to cancers
Humira (Adalimumab)
Human antibodies that bind TNF-α. Used to treat rheumatoid arthritis, Crohn’s Disease, plaque psoriasis, psoriatic arthritis, ankylosing spondylitis, Juvenile Idiopathic Arthritis
Praluent (Alirocumab)
Human antibodies that bind PCSK9. PCSK9 induces LDL-R degradation when someone has high circulating LDL. Lowers cholesterol
Reopro mechanism
Platelets express an integrin known as GP2b/3a, which binds to fibrinogen. Allows the platelets to come together to form a “white clot”. Reopro acts against the GP2b/3a integrin on the platelet surface. Prevents the platelets from clumping together and forming a blood clot
Avastin mechanism
An anti-cancer drug that is against angiogenesis (development of new blood vessels to bring nutrients to a tumor). Based on vascular endothelial growth factor A (VEGF-A). When VEGF-A binds to its receptor on endothelial cells, it promotes angiogenesis. Avastin is an antibody against VEGF. It prevents it from binding to its receptor and therefore prevents angiogenesis
Humira mechanism
An anti-TNF antibody. When TNF binds to its receptor, it activates pro-inflammatory pathways that lead off with TAB1 and TAB2 binding proteins. These will create signals that will lead to eventual activation of NFKB and the transcription of pro-survival and pro-inflammatory cytokines. Humira binds to TNF, so it can’t bind to its receptor, and NFKB is not activated
Praluent mechanism
LDL is taken from the blood into the cells by LDL receptors. PCSK9 acts as a co-receptor. When PCSK9 binds to LDL receptors, it causes the receptors to degrade and prevents LDL from being taken up by the cell. Praluent binds to PCSK9 so it can’t bind to the LDL receptor and cause it to degrade. More LDL is endocytosed into the cell, causing a decrease in circulating LDL
How is class switching activated?
A naive B cell’s MHC molecule binds to the TCR on an activated TH2 cell. The activated TH2 cell secretes IL-4 and IL-5, which allows somatic hypermutation and class switching to occur
