Abstract
Receptor tyrosine kinase-like orphan receptor 2 (ROR2) has been identified as a highly relevant tumor-associated antigen in a variety of cancer indications of high unmet medical need, including renal cell carcinoma and osteosarcoma, making it an attractive target for targeted cancer therapy. Here, we describe the de novo discovery of fully human ROR2-specific antibodies and potent antibody drug conjugates (ADCs) derived thereof by combining antibody discovery from immune libraries of human immunoglobulin transgenic animals using the Transpo-mAb mammalian cell-based IgG display platform with functional screening for internalizing antibodies using a secondary ADC assay. The discovery strategy entailed immunization of transgenic mice with the cancer antigen ROR2, harboring transgenic IgH and IgL chain gene loci with limited number of fully human V, D, and J gene segments. This was followed by recovering antibody repertoires from the immunized animals, expressing and screening them as full-length human IgG libraries by transposon-mediated display in progenitor B lymphocytes (“Transpo-mAb Display”) for ROR2 binding. Individual cellular “Transpo-mAb” clones isolated by single cell sorting and capable of expressing membrane-bound as well as secreted human IgG were directly screened during antibody discovery, not only for high affinity binding to human ROR2, but also functionally as ADCs using a cytotoxicity assay with a secondary anti-human IgG-toxin-conjugate. Using this strategy, we identified and validated 12 fully human, monoclonal anti-human ROR2 antibodies with nanomolar affinities that are highly potent as ADCs and could be promising candidates for the therapy of human cancer. The screening for functional and internalizing antibodies during the early phase of antibody discovery demonstrates the utility of the mammalian cell-based Transpo-mAb Display platform to select for functional binders and as a powerful tool to improve the efficiency for the development of therapeutically relevant ADCs.
Highlights
Cancer is still a leading cause of death worldwide
Immunoglobulin transgenic mice expressing antibodies with fully human VH and VL sequences, provided by Harbor BioMed, Cambridge, MA, were immunized with the extracellular domain of human receptor 2 (ROR2) containing a C-terminal Twin-Strep-tag. hROR2-specific B lymphocytes were enriched from the spleens of immunized mice using magnetic activated cell sorting (MACS), RNA was isolated from hROR2-ECD-TwinStrep enriched B lymphocytes and libraries of coding regions for human VH and kappa VL variable domains were amplified by RTPCR using specific primers
Following DNA library construction, cellular libraries were generated by stable transposition of heavy chain (HC) and light chain (LC) transposable vectors to stably display fully human IgG1 antibodies on the surface of immortalized murine L11 pro-B cells derived by subcloning from the A-MuLV transformed RAG-2 knock-out cell line 63-12 [36], unable to express endogenous murine Ig components
Summary
Cancer is still a leading cause of death worldwide. While the landscape of cancer treatment has positively evolved recently with the advent of targeted, including antibody- and cellbased therapies, broadly effective and curative treatment options still remain limited. Naked antibodies either need to have an intrinsic capability to interfere with the growth of cancer cells, or they need to recruit other immune-system components to inhibit tumor growth and expansion. These activities of antibodies are often insufficient to effect complete eradication of targeted tumor cells. In order to generate potent, and safe ADCs, many aspects of the molecule need to be optimal This includes the tumorselective binding of the antibody moiety to the targeted cancer cells, and functional properties of the antibody moiety, such as its ability to internalize the ADC into the targeted cancer cell following binding of the ADC to the TAA. In the field of targeted cancer therapies, ADCs will have an enormous potential for cancer treatment in the future, if engineered diligently with regard to linker stability and product homogeneity as well as optimal tumor selectivity and functionality
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