Thinking back to the discussion on “Hybridoma Development”, the initiation of any antibody program (monoclonal or polyclonal) begins with a series of immunizations in order to generate a specific antibody response. How the immune system responds to a specific antigen will determine the success or failure of any given project. Therefore, it is important for us to understand and consider the fundamentals of the immune system to try to drive appropriate responses and maximize the likelihood of success.
II. Fundamentals of the Immune Response
The immune system is our primary defense mechanism made up of barriers, cells, and circulating proteins. Barriers such as the skin are the first line of defense by keeping invading microbes out, however if the barrier is broken or penetrated (Figure 1, step 1) the components of the innate immune system kick in and an immune response is initiated (Figure 1, step 2). Macrophages and dendritic cells become activated through the stimulation of pattern recognition receptors (Toll-like Receptors, NOD-like receptors, C-type lectin receptors, etc.) that detect the presence of “foreign” material through the identification of pathogen-associated molecular patterns (lipopolysaccharides, bacterial cell wall components, nucleic acids, microbial proteins, etc.) that are found on bacteria, fungi, viruses, and parasites. The activation of the macrophages and dendritic cells will lead to phagocytosis of the invading microbe and secretion of proinflammatory cytokines and chemokines. This initial innate response serves to begin to detect and remove the invader and also prevent the spread of the invader systemically. It also will recruit other immune cells to the fight, such as neutrophils, and mobilize macrophages and dendritic cells to migrate to the nearest lymph node (Figure 1, step 3). In the local or draining lymph node, the B cells and T cells get exposed to the antigens of the invader, primarily through the presentation from the dendritic cells and macrophages (Figure 1, step 3). Those B cells and T cells that are activated through the recognition of antigen, begin to divide and interact with one another. This will initiate the adaptive immune response through the generation of antibody and T cell responses that are specific to the microbe. The antibodies produced by the B cells are then released into circulation (Figure 1, step 4) to direct the immune system to the specific pathogen and allow for the capture and clearance of the pathogen. This process will develop specifically to the pathogen. Memory B cells and T cells will develop to be able to protect the host against future exposures to the same or related microbe. Depending on the site of infection and the amount of the pathogen that is present, this will drive the specific type of response necessary for efficient detection, capture, and removal.
III. Considerations for Immunization
The purpose of an immunization, whether it be for an antibody development project or a vaccination, is to develop a specific response to the immunogen (the material that is used to generate an immune response). Understanding the fundamentals of the immune response provides insight into best practices for immunization protocols during the hybridoma development process. Essentially, in a hybridoma development project, we want to mimic an immune response, which includes the initiation of the inflammatory response by the innate immune system. To do this, we use an adjuvant mixed with the immunogen. The adjuvant contains compounds that stimulate specific pattern recognition receptors and initiate an inflammatory response, which will activate innate immunity and drive an immune response. While the adjuvant will initiate the response, the immunogen will be the target of the response.
While there are a number of immunization strategies, we must keep in mind that the route, dose, and exposure period all are key elements to an immune response. The route of exposure can play a role in how the immune system responds. The most common routes are subcutaneous (sc) and intraperitoneal (ip). The sc immunization will generate a more localized response while the ip provides a more systemic exposure. The exposure duration is important in maturing the response to be specific for the immunogen. This generally requires several immunizations. The dose will also play a role in generating the specificity to the immunogen. In some cases, the dose may be modulated to drive affinity maturation to produce antibodies with higher specificity. In cases where an antibody response is difficult to generate for a specific immunogen; changing the route, dose, and/or adjuvant may help to engage the immune system differently to produce a more robust response.
Depending on the overall goals of the hybridoma project, different adjuvants and routes can be used to tune the immune response and increase antibody diversity and affinity. It is important prior to the initiation of the project to consider the best options for each specific immunogen and analyse the response to guide decisions, particularly in the case of difficult immunogens.
ABOUT DR. PELSUE
Dr. Pelsue joined MBS as Science Director after 20 years experience in Immunology research. Dr. Pelsue is providing technical expertise and leadership to address challenging new antibody targets on behalf of our client base.