Highly reliable GIGA-sized synthetic human therapeutic antibody library construction.

Background: Monoclonal antibodies (mAbs) and their derivatives are the fastest expanding category of pharmaceuticals. Efficient screening and generation of appropriate therapeutic human antibodies are important and urgent issues in the field of medicine. The successful in vitro biopanning method for antibody screening largely depends on the highly diverse, reliable and humanized CDR library. To rapidly obtain potent human antibodies, we designed and constructed a highly diverse synthetic human single-chain variable fragment (scFv) antibody library greater than a giga in size by phage display. Herein, the novel TIM-3-neutralizing antibodies with immunomodulatory functions derived from this library serve as an example to demonstrate the library's potential for biomedical applications. Methods: The library was designed with high stability scaffolds and six complementarity determining regions (CDRs) tailored to mimic human composition. The engineered antibody sequences were optimized for codon usage and subjected to synthesis. The six CDRs with variable length CDR-H3s were individually subjected to ß-lactamase selection and then recombined for library construction. Five therapeutic target antigens were used for human antibody generation via phage library biopanning. TIM-3 antibody activity was verified by immunoactivity assays. Results: We have designed and constructed a highly diverse synthetic human scFv library named DSyn-1 (DCB Synthetic-1) containing 2.5 × 1010 phage clones. Three selected TIM-3-recognizing antibodies DCBT3-4, DCBT3-19, and DCBT3-22 showed significant inhibition activity by TIM-3 reporter assays at nanomolar ranges and binding affinities in sub-nanomolar ranges. Furthermore, clone DCBT3-22 was exceptionally superior with good physicochemical property and a purity of more than 98% without aggregation. Conclusion: The promising results illustrate not only the potential of the DSyn-1 library for biomedical research applications, but also the therapeutic potential of the three novel fully human TIM-3-neutralizing antibodies.

Generation of Potent Anti-Vascular Endothelial Growth Factor Neutralizing Antibodies from Mouse Phage Display Library for Cancer Therapy.

Vascular endothelial growth factor (VEGF) is an important stimulator for angiogenesis in solid tumors. Blocking VEGF activity is an effective therapeutic strategy to inhibit tumor growth and metastasis. Avastin, a humanized monoclonal antibody recognizes VEGF, has been approved by the US Food and Drug Administration. To generate potential VEGF-recognizing antibodies with better tumor regression ability than that of Avastin, we have designed a systematic antibody selection plan. From mice immunized with recombinant human VEGF, we generated three phage display libraries, scFv-M13KO7, Fab-M13KO7, and scFv-Hyperphage, in single-chain Fv (scFv) or Fab format, displayed using either M13KO7 helper phage or Hyperphage. Solid-phase and solution-phase selection strategies were then applied to each library, generating six panning combinations. A total of sixty-four antibodies recognizing VEGF were obtained. Based on the results of epitope mapping, binding affinity, and biological functions in tumor inhibition, eight antibodies were chosen to examine their abilities in tumor regression in a mouse xenograft model using human COLO 205 cancer cells. Three of them showed improvement in the inhibition of tumor growth (328%-347% tumor growth ratio (% of Day 0 tumor volume) on Day 21 vs. 435% with Avastin). This finding suggests a potential use of these three antibodies for VEGF-targeted therapy.

Role of IL-6 in neuroendocrine differentiation and chemosensitivity of non-small cell lung cancer.

Interleukin-6 (IL-6) has been shown to regulate both growth and neuroendocrine (NE) differentiation in some types of human cancer cells, and erbB2 may be a critical component of IL-6 signaling. Non-small cell lung cancer (NSCLC) tumors that demonstrate NE properties have been suggested to have biological characteristics similar to small cell lung cancers with initial responsiveness to chemotherapy. We investigated whether IL-6 is implicated in the cell growth, NE differentiation, and chemosensitivity of NSCLC-NE cells. NSCLC-NE cells were treated with exogenous IL-6, and a subclone of an IL-6-transfected NSCLC cell line that constitutively expressed IL-6 receptor was also generated. These cells were assessed for cell proliferation by cell counting and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays, chemosensitivity to cisplatin and etoposide by MTT assays, and NE differentiation by observing morphological changes and immunoblotting for neuron-specific enolase (NSE). The IL-6-treated cells and the IL-6-transfected cells showed enhanced cell proliferation and downregulated NSE expression, but little change in chemosensitivity. In the culture medium, IL-6-transfected cells grew as looser aggregates than the parental cells. IL-6 could not activate the erbB genes. In conclusion, IL-6 can induce cell proliferation and NE dedifferentiation but has little effect on chemosensitivity in IL-6 receptor-expressing NSCLC-NE cells. The status of NSE expression is unlikely to be a crucial factor for chemosensitivity in NSCLC cells.