Strategies for generating therapeutic antibodies
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Monoclonal antibodies have become essential therapeutic tools and currently dominate the therapeutic protein market. Consequently, there is continued demand for new therapeutic antibodies and their discovery techniques. In one part of this work, we report the discovery of a new therapeutic antibody candidate with a novel mechanism for inhibition of a therapeutically relevant biochemical pathway: the classical complement pathway. In order to inhibit classical complement, an antibody was developed that modulates the signaling subcomponent of the pathway initiating C1 complex, C1s. This work includes novel protocols and strategies used for discovery and characterization of antibody D, which binds and inhibits C1s protease activity. By regulating C1s activity, antibody D is shown to regulate classical complement. It is further shown that affinity maturation of antibody D results in higher levels of complement inhibition at various antibody concentrations. This work marks the first example of an antibody that specifically regulates the classical complement pathway by targeting the C1s protease on the pathway initiating C1-complex. Next, we characterize the human immune cells produced by humanized NSG mice, most notably the B and T lymphocytes, engraftment with human CD34+ HSC cells. We detected development of naïve human B and T cells and their various subtypes, as well as other human immune cells from engrafted mice. However, attempts to generate a robust antibody response to antigens were unsuccessful. Therefore, we conclude that NSG humanized mice developed in this study are suitable for studying the antibody repertoire of naïve B cells, however they are not suitable for the analysis of activated B-cells. Last, we introduce a novel strategy for the generation of polarized antibody repertoires for use in therapeutic monoclonal antibody discovery. This technique combines targeted antigen delivery to a specific lymph node and a frequency based antibody selection approach in order to directly select antigen specific antibodies in silico. By directly selecting antigen-specific antibodies, this approach circumvents laborious and time consuming screening techniques. We expect that this work will be the foundation of an overall improved protocol for monoclonal antibody discovery that accelerates the speed and enhances the simplicity of discovery techniques.