Top strategies for therapeutic antibody discovery
Antibodies have become a leading therapeutic for treating many diseases. Not surprisingly, the marketplace for antibodies (and biologics in general) is expected to grow exponentially to $300 Billion USD by 2025. With the increased success of antibody therapeutics in the clinic, a number of strategies have arisen to streamline discovery. Discovery technologies can be divided into three main categories: Transgenic animals, display libraries and donor B cell sorting. Although vastly different, all three technologies have successfully yielded therapeutic antibodies. Transgenic animals led to the development of panitumumab, an anti-EGFR antibody approved by the US FDA in 2006. Display libraries led to the development of adalimumab, an anti-TNF-alpha antibody approved in 2002. Donor B cell sorting led to the development of bamlanivimab, an anti- SARS-CoV-2 spike protein antibody approved for emergency use authorization in 2021.
The use of transgenic animals was first reported with mice and led to the development of the HuMabMouse and the XenoMouse. Transgenic animals are generated by inserting human immunoglobulin genes into the animal genome. The transgenic animals are immunized with the target of interest to promote production of antibodies against the target. Resulting B-cells from immunized animals are isolated for hybridoma generation. Hybridomas are screened for binding against the target of interest. The primary advantage of a humanized mouse is that antibodies are discovered in a human framework. Prior to transgene engineering, processes such as CDR grafting were required to convert a mouse or rodent antibody into a human antibody. Implementation of transgenic animals no longer requires humanization of antibodies discovered from immunized animals.
Display libraries are a collection of single chain variable fragments (scFv) or fragment antigen binding (Fab) that are expressed on the cell surface of phages, yeast or mammalian cells. This collection of scFv or Fab sequences may be fully synthetic, isolated from natural repertoires or semi-synthetic (modified derivatives or sub-selection of natural antibody repertoires). For each display organism, the scFv/Fab libraries are fused to a naturally occuring membrane protein. The specific membrane anchor used is dependent on the organism of choice. In phage display, phage coat proteins are commonly used as the membrane anchor. In yeast display, the protein Aga2 is commonly used as the membrane anchor, whereas in mammalian display, the commonly used membrane anchor is the transmembrane region of PDGFR.. While all three systems are able to display both scFv and Fab, mammalian cells have the added ability to display full IgG antibodies. The three systems additionally differ in the diversity of library sequences that can be screened in a single campaign. Phage display libraries have a diversity bottleneck of 10e11 to 10e12, yeast display libraries have a diversity bottleneck of 10e7 to 10e8 and mammalian display libraries have a diversity bottleneck of 10e5 to 10e6.
Single B cell isolation is a novel technology that takes advantage of the human immune system. B cells from healthy or patient blood cell donors are screened for binding against a target of interest. Both healthy and patient donors provide a natural human antibody repertoire that have undergone self-target counterselection and non-stable sequence depletion. When developing antibodies against a particular disease, patient donors may have an enrichment of antibodies against a disease relevant target.
In all of the aforementioned discovery processes, CDR sequences from the top performing antibodies will be grafted onto a full human IgG format. The lead molecules will have to be tested for biological activity and manufacturability prior to scaling up production and clinical trials.