Wild-type (WT) C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were immunized with purified human C6 protein (Complement Technology, Inc.). Hybridomas were developed according to an established protocol.12 Clone 1C9 was identified by conventional ELISA screening of the supernatants of resultant hybridoma cultures. All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of Cleveland Clinic.
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A method has been developed for the rapid generation of high-affinity humanized antibodies from immunized animals without the need to make conventional hybridomas. Rearranged IgH D(J) regions were amplified from the spleen and lymph tissue of mice immunized with the human complement protein C5, fused with a limited repertoire of human germline heavy chain V-genes to form intact humanized heavy chains, and paired with a human light chain library. Completed heavy and light chains were assembled for mammalian cell surface display and transfected into HEK 293 cells co-expressing activation-induced cytidine deaminase (AID). Numerous clones were isolated by fluorescence-activated cell sorting, and affinity maturation, initiated by AID, resulted in the rapid evolution of high affinity, functional antibodies. This approach enables the efficient sampling of an immune repertoire and the direct selection and maturation of high-affinity, humanized IgGs.
Competing interests: The authors have the following interests. AnaptysBio, Inc. funded the research described in this report. AnaptysBio encourages publication and plays a role in issuing approval for the authors to submit scientific manuscripts to journals. All authors are employed by AnaptysBio, Inc. and the technology described in this paper is a product of AnaptysBio ( ). There are no further patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.
Despite these innovations, novel approaches to therapeutic antibody development are still needed to address limitations associated with wild-type and transgenic animal immunization methods. The generation and screening of hybridomas from an immunized animal is time-consuming and samples only a fraction of the antibodies generated during the adaptive immune response. In addition, mAb humanization can be challenging when heavy and light chain variable (V) regions have limited homology to the closest human V-region, requiring iterative rounds of engineering and maturation to reach design metrics. Even when humanization is successful, the resulting antibody may still trigger an immune response. An approach that minimizes the non-human sequence incorporated into the final antibody without sacrificing functionality would therefore be useful.
In this study we demonstrate an alternative approach to antibody discovery that combines mouse immunization with rapid humanization using mammalian cell surface display and AID-induced affinity maturation. This method obviates the need to isolate and screen individual hybridomas by generating a humanized antibody library directly from immunized mice. The rearranged heavy chain D(J) regions from mice immunized with the antigen C5-C345C antigen were PCR amplified from spleen and lymph node cDNA, and incorporated into synthesized germline IGHV regions. This HC library was combined with a human LC library [21], yielding humanized antibodies enriched for potential antigen binders. Transfection of this library into HEK 293 cells together with AID, and in a format compatible with surface display, facilitated the FACS selection of numerous C5-C345C specific antibodies. One of these clones was selected for subsequent affinity maturation to a KD of 200 pM, and was demonstrated to possess a potent, dose-dependent ability to inhibit MAC formation and complement-mediated cell lysis.
This method couples the robust humoral adaptive immune response with maturation by AID-induced SHM, mimicking the critical features of adaptive immunity in vitro to produce high affinity antibodies. Incorporating HC D(J) regions from immunized mice into the displayed library provides greater potential to recover high affinity, efficacious antibodies for the antigen of interest relative to HC D(J) diversity derived from unselected, antigen-naïve sources. This approach also minimizes the amount of mouse sequence incorporated into the final antibody, in contrast to standard hybridoma and humanization strategies. We recovered eight unique HC/LC pairs from initial discovery, and anticipate that this method could be scaled to provide additional diversity. The subsequent application of mammalian display in combination with SHM allows selection pressure to be applied to evolving populations of antibodies such that they may be driven to desired end points including high affinity and specificity, and also other important parameters such as stability and high expression levels in mammalian cells. APE1224, composed of V-regions IGHV3-23 and IGKV3-20, was typical of antibodies isolated using this approach, expressing well in HEK 293 cells and unfolding with a Tm of 78C, indicative of high thermostability. This method is potentially applicable to antibodies from any origin, and although demonstrated here through active immunization of mice, it may equally well be applied to harvesting antibodies from the immune repertoire of other species, including humans.
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