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.

Dengue virus induces expression of CXC chemokine ligand 10/IFN-gamma-inducible protein 10, which competitively inhibits viral binding to cell surface heparan sulfate.

Dengue virus is an arthropod-borne flavivirus that causes a mild febrile illness, dengue fever, or a potentially fatal syndrome, dengue hemorrhagic fever/dengue shock syndrome. Chemokines primarily orchestrate leukocyte recruitment to the areas of viral infection, which makes them critical mediators of immune and inflammatory responses. In the present study, we investigated the induction and function of chemokines in mice early after infection with dengue virus in vivo. We found that CXCL10/IFN-gamma-inducible protein 10 (IP-10) expression was rapidly and transiently induced in liver following infection. The expressed CXCL10/IP-10 likely mediates the recruitment of activated NK cells, given that anti-CXCL10/IP-10-treated mice showed diminished NK cell infiltration and reduced hepatic expression of effector molecules in activated NK cells after dengue virus infection. Of particular interest, we found that CXCL10/IP-10 also was able to inhibit viral binding to target cells in vitro. Further investigation revealed that various CXCL10/IP-10 mutants, in which the residues that mediate the interaction between the chemokine and heparan sulfate were substituted, failed to exert the inhibitory effect on dengue binding, which suggests that CXCL10/IP-10 competes with dengue virus for binding to heparan sulfate on the cell surface. Moreover, subsequent plaque assays showed that this inhibition of dengue binding blocked viral uptake and replication. The inhibitory effect of CXCL10/IP-10 on the binding of dengue virus to cells may represent a novel contribution of this chemokine to the host defense against viral infection.

Both CXCR3 and CXCL10/IFN-inducible protein 10 are required for resistance to primary infection by dengue virus.

We examined the extent to which CXCR3 mediates resistance to dengue infection. Following intracerebral infection with dengue virus, CXCR3-deficient (CXCR3(-/-)) mice showed significantly higher mortality rates than wild-type (WT) mice; moreover, surviving CXCR3(-/-) mice, but not WT mice, often developed severe hind-limb paralysis. The brains of CXCR3(-/-) mice showed higher viral loads than those of WT mice, and quantitative analysis using real-time PCR, flow cytometry, and immunohistochemistry revealed fewer T cells, CD8(+) T cells in particular, in the brains of CXCR3(-/-) mice. This suggests that recruitment of effector T cells to sites of dengue infection was diminished in CXCR3(-/-) mice, which impaired elimination of the virus from the brain and thus increased the likelihood of paralysis and/or death. These results indicate that CXCR3 plays a protective rather than an immunopathological role in dengue virus infection. In studies to identify critical CXCR3 ligands, CXCL10/IFN-inducible protein 10-deficient (CXCL10/IP-10(-/-)) mice infected with dengue virus showed a higher mortality rate than that of the CXCR3(-/-) mice. Although CXCL10/IP-10, CXCL9/monokine induced by IFN-gamma, and CXCL11/IFN-inducible T cell alpha chemoattractant share a single receptor and all three of these chemokines are induced by dengue virus infection, the latter two could not compensate for the absence of CXCL10/IP-10 in this in vivo model. Our results suggest that both CXCR3 and CXCL10/IP-10 contribute to resistance against primary dengue virus infection and that chemokines that are indistinguishable in in vitro assays differ in their activities in vivo.

Reduced 2,4-dinitro-1-fluorobenzene-induced contact hypersensitivity response in IL-15 receptor alpha-deficient mice correlates with diminished CCL5/RANTES and CXCL10/IP-10 expression.

Using a model of 2,4-dinitro-1-fluorobenzene-induced contact hypersensitivity (CHS) we found that, as compared with wild-type mice, IL-15 receptor alpha chain (IL-15Ralpha)-deficient mice showed significantly less ear swelling. This decreased response was associated with diminished expression of CCL5/RANTES and CXCL10/IP-10, chemokines critical for effector cell recruitment, in the inflamed tissue. We determined that both the number of CD8(+) T cells infiltrating the affected skin and the production of CCL5/RANTES by antigen-stimulated CD8(+) T cells were decreased in IL-15Ralpha(-/-) mice. The lower levels of CXCL10/IP-10 suggested that the IL-15Ralpha(-/-) mice had reduced production of IFN-gamma, the primary inducer of CXCL10/IP-10, which was in fact the case. However, by contrast with CCL5/RANTES, the diminished levels of IFN-gamma were likely due to the decreased number of skin-infiltrating CD8(+) T cells, since IFN-gamma production by antigen-stimulated CD8(+) T cells was comparable between wild-type and IL-15Ralpha(-/-) mice. Our data suggest a positive, pro-inflammatory feedback loop involving CCL5/RANTES, IFN-gamma and CXCL10/IP-10 that underlies the CHS reaction and that is disrupted, likely primarily by a defect in CCL5/RANTES production, in mice lacking IL-15Ralpha, resulting in impaired leukocyte recruitment and inflammation. Moreover, it is particularly noteworthy that the defect in CCL5/RANTES expression in CD8(+) T cells is intrinsic to the absence of IL-15Ralpha, indicating that IL-15Ralpha is critical for CCL5/RANTES expression in CD8(+) T cells.