Antibodies are glycoproteins that specifically recognize antigen. They’re produced by cells of the immune system - called B cells or plasmocytes - in order to block and neutralize foreign agents while infection occurs. In case of auto-immunity diseases (e.g. diabetes, lupus…) antibodies are secreted against host molecules, and are so named auto-antibodies.

Antibodies are part of the immunoglobulin family, composed by 4 chains of amino-acids : two light chains and two heavy chains, shaping a Y letter. Each light chain consists of a constant domain and a variable domain; heavy chains are composed of a variable fragment and three or four constant fragments according to the isotype. For a given antibody, the two heavy chains are identical, likewise for the two light chains. 

The isotype of an antibody refers to its class. In fact, antibodies are divided in class and subclasses depending on their structure of their heavy chain.

A different structure of an antibody affects directly its valence, its means the number of epitopes the antibody is able to recognize. The γ, α, μ, ε and δ chains correspond respectively to IgG, IgA, IgM, IgE and IgD immunoglobulins. There are also subclasses of immunoglobulins, reflecting finer differences between heavy chains, and being radically different for each species.

The human thus has four subclasses of IgG and two subclasses of IgA, whereas rat and guinea pig species have only two subclasses of IgG, and rabbit present only one subclass of IgG but fourteen IgA subtypes! There are also light chain isotypes, which can be κ (kappa) or λ (lambda).

Antibodies are encoded by V, D and J genes undergoing recombination in B cells. This recombination is, in association with the phenomena of somatic hypermutation and junctional variability, the source of their diversity.

The B cell leaves the bone marrow without ever having seen the epitope of its antigen: the specificity and the affinity of the membrane antibody, and consequently of the circulating antibody produced, are determined before the encounter. To adjust these parameters to the antigen initiating a humoral mediated immune response, the activated B cell clone will initiate somatic hypermutation: the genetic recombination at the origin of the immunoglobulin produced will undergo mutations in its sequence which will adjust the hypervariable regions to the epitope. Mutations take place on the variable segments of the light and heavy chains.

A monoclonal antibody recognizes only one epitope of a given antigen and comes from only one single B clone. The polyclonal antibodies are a mixture of antibodies recognizing different epitopes on a given antigen, each idiotype being secreted by a different plasmocyte. During the immune response, an organism synthesizes antibodies directed against several epitopes of an antigen: the answer is called polyclonal. In vivo, meaning in animal, the response is always polyclonal.

Producing monoclonal antibodies in vitro has long been difficult, because of the short life of antibody-secreting cells, the B cells. The antibodies were then obtained in vivo by injecting the animal with a given antigen and collecting the antibodies in its blood or ascites after intraperitoneal injection. This method gave antibodies but polluted by many impurities.

In the late 1970s, César Milstein and Georges Köhler developed the hybridoma technique in mouse, while Hervé Bazin developed the hybridoma technique in rat species thanks to unique proprietary myeloma cell line. Hervé Bazin is at the origin of SYnAbs company.

The antigen is injected into the animal, and spleen cells are removed after a few weeks. In these cells are plasmocytes secreting antibodies directed specifically against the selected antigen. These plasma cells are then fused in vitro with myelomas (or fusion partners) - by electrofusion or PEG adding - which are tumor cells with the property of multiplying indefinitely.

The easiest system for expression of mammalian proteins remains the human embryo kidney cell line 293 (HEK 293). Despite the fact that creation of high producer expression system has been continuously improved, it is still too time-consuming and expensive for research applications, or if large numbers of individual antibodies have to be produced for screening purposes.

In this case, transient antibody expression in HEK cell line is much more suitable because it allows fast and parallelized production without any need to generate stable producer cell lines. We add that transient mammalian antibody production can be easily scaled up by employing batch or fed-batch mode during UpStream process and finally generate grams of desired monoclonal antibodies or recombinant proteins.

While hybridoma cell lines are sufficient for producing small amounts of monoclonal antibodies, a lot of limitations arise when we start speaking about large-scale commercial manufacturing.

When evaluating possible expression systems for monoclonal antibodies production, several factors need to be taken into account. First, they need to easily be genetically modified in order to ensure high productivity capacity and high success rate during stable transfection step. Then, these host cell lines have to confer the proper post-translational modifications of folded recombinant proteins. At last, the chosen expression system has to be safe and not susceptible to adventitious agents.

Through many years of experimentations with CHO cell lines since 1919, it appears they met several of these requirements, also with the ability of fast cell culture scaling-up.

The first product produced in CHO cells, approved in 1987, was a recombinant therapeutic protein manufactured by Genentech called Activase ; the yields were around 100 mg/liter and the media in which the cells grew contained animal products, such as fetal bovine or fetal calf serum.

A classical project for the generation of a new monoclonal antibody will take an average of three to four months.

After an in-depth analysis of the targeted antigen, SYnAbs start the project with the generation of the antigen if needed, or the subcontracting of chemical synthesis or small peptide generation with our partner. The customer can also provide the antigen if this option is chosen.

Depending of the antigen, SYnAbs offers different species (mouse, rat, guinea pig) and immunization routes. After injection, the immune serum will be tested at day 30 with an ELISA and the best-immunized animal will receive a third immunization and the fusion will be done with this animal after 8 weeks.

Greg Winter's team demonstrated in 1989 that it was possible to obtain affinities of the order of ten nanomolar using only the variable domains (V) VH (H for heavy chain) of mouse antibodies obtained by in vitro selection from immunized animals. The authors created for the occasion the term dAb for single domain antibodies. They already mentioned the property of these dAb to target epitopes inaccessible to conventional antibodies. This discovery remained anecdotal until 1993 when a team demonstrated the presence of conventional antibodies but also antibodies devoid of light chains and CH1 domains (C for constant domain) in the serum of camels. These antibodies called HcAb for heavy chain only antibodies bind their targets by a single variable domain named VHH to differentiate it from conventional VH. 

By reference to their small size, camel dAb, considered as the smallest active antibody fragments, are sometimes called nanobodies. It should be noted that all camelids, including camels, llamas and vicuñas, express this type of antibody. In parallel, similar antibodies have also been found in some types of sharks. They are then called Ig-NAR (for new antigen receptor) and their only variable domain is called V-NAR.

Subsequently, several teams have developed antibody engineering approaches that first partially confer the properties of lama dAb to human VH (camelization) by substitution of key residues, then finally select in vitro from large libraries of human VH or VL (L for Light Chain) optimized domains, human dAb whose stability and expression performance properties were close to those of natural dAbs.