Intraperitoneal (IP) and intravenous (IV) injections are well-established immunization procedures for raising mouse antibodies, and are considered as gold standard. But in the particular case of poor immunogenic compounds or haptens (like peptides, polysaccharides, lipids, small molecules…), traditional strategies can fail to generate large panels of mAbs and finally get a monoclonal antibody of high affinity and great specificity.

SYnAbs has found the solution to handle this difficult situation. Injection into footpad and the work on popliteal ganglia breaks the immunotolerance and triggers an immune response that would not normally occur in the animal. The antigenic presentation by activated resident APCs at the secondary lymphoid organs doesn’t occur in the same way as in the spleen area.

Once primed, dendritic cells migrate to the draining lymph nodes, the DC population becoming more and more complex as migration progresses. It turns out that additional dendritic cell subpopulations appear during the process - whose phenotypes are non-existent in the spleen - and which interact differently with the T and B lymphocytes during the creation of the adaptive immune response. This latter route provides stronger stimulation, and results in a higher magnitude of the immune response. This may help reveal the subdominant epitopes, but does not seem to change the epitope hierarchy.

« So if it works so well, what is the purpose of DNA immunization ? » you should ask.

DNA immunization solves different issues when you face the following situations : 

• Raising antibodies against difficult to express proteins, such as disulfide-rich domains or multi-spanning membrane proteins (MPs). If the proteins are naturally expressed in a membrane associated format, such as the multi-transmembrane G-protein coupled receptors (GPCRs) and ion channels, traditional approaches have difficulty producing full-length protein immunogens by the recombinant protein method,

• Generating monoclonal antibodies against expensive antigens, when recombinant protein is limiting, due to the price to produce or purchase,

• Raising antibodies against intracellular proteins : one may assume that it is necessary to re-direct intracellular proteins into secretory pathways by adding a signal peptide to elicit a better antibody response. However, in a number of monoclonal antibody production studies, DNA has been successfully used as immunogen.

• Generating mAbs triggering bacteria, virus antigens or toxins, for which bioproduction in classical expression systems is problematic and the injection potentially lethal for the animal,

• Raising antibodies targeting conformational antigens : peptides often do not accurately mimic the native conformation of a targeted protein, and peptide immunization often leads to the unpleasant surprise of not recognizing native protein during screening step. You can choose to develop different delivery systems including whole cells, membrane fractions, or membrane-derived vesicles, which retain the protein in the native membrane environment. However, the epitope typically represents only a small fraction of the total protein and thus, a large non-specific antibody response is often observed for these formats. Consequently, extensive counter-screening using multiple different cell lines is required, significantly expanding the cost and time for antibody generation.

DNA immunization as it exists today was pioneered in the early 1990s. Its initial use as a vaccination platform generated great excitement due to the overall simplicity of using DNA plasmids to deliver immunogens. One particularly attractive feature of DNA vaccines is that immunogens are produced in vivo, giving them the ability to induce T-cell immune responses through endogenous antigen processing and presentation pathways.

But aside this last consideration, DNA immunization present many advantages :

• Relative simplicity of manipulation and production, no antigen is needed. Antigens are sometimes difficult to express or purify and to find in catalogs because they are toxic, insoluble or unstable. When produced in recombinant way, it can be different from the native form and therefore lead to irrelevant antibodies. You consequently save some precious time and costs. There’s also no risk to inject contaminant (results of antigen production) like co-purified proteins, protein purification tags or Host Cell Proteins.

• More useful than traditional approaches to generating mAbs against more difficult targets, especially membrane proteins. The DNA immunization approach can circumvent these problems because full-length proteins can be expressed in vivo when they are delivered in the form of genetic immunogen.

• Effective in generating mAbs against conformation sensitive targets. It is well known that the structural integrity of proteins is critical for the induction of functional mAbs, yet these sensitive structures tend to be lost during the in vitro protein production process, regardless of whether they are produced as recombinant proteins or are extracted directly from cells. Production of functionally active mAbs is highly dependent on the conformation of the proteins. Expressing intact immunogens in vivo by DNA immunization appears to have the best chance of inducing mAbs with the desired biological activities.

• DNA immunization does not require the production or purification of proteins from a pathogen, which avoids any concerns related to biosafety. 

• As a simple and flexible immunogen design approach, DNA immunization offers a wide range of options to produce novel immunogen inserts. Antigen can be full length, sub-units, fragments, novel formats, fusion proteins...

• One unique feature of DNA immunization is the convenience of using the same DNA vaccine constructs to express antigens and for mAb screening step. Cell-associated antigen-based screening has been widely and successfully used for mAbs targeting transmembrane proteins, viral envelope proteins and intracellular proteins. In these cases, cells expressing the immunogens are used without the need for protein purification to screen the binding activity of mAbs by either fluorescence-activated cell sorting analysis, whole cell enzyme-linked immunosorbent assay (ELISA), or immunohistochemistry (IHC) methods.

• DNA prime-protein boost combo. One approach that presents a great advantage for the induction of high-titer and high-quality antibody responses is the heterologous prime-boost approach. In this approach, the DNA vaccine is delivered as the priming immunization, followed by a boost with protein antigens as recombinant proteins or peptides. One finding regarding DNA priming immunization is their ability to induce higher-level of antigen specific B-cell responses.

SYnAbs has the capacity to offer DNA immunization to its partners to generate innovative monoclonal antibodies against transmembrane proteins, expensive and difficult-to-produce antigens, conformational epitopes and many other complex antigens to target thanks to its own DNA plasmid construct.