Rapid isolation of anti-idiotype aptamers for quantification of human monoclonal antibodies against SARS-CoV-2 spike protein.

Therapeutic antibodies that block viral entry have already proven to be important, first line drugs for treatments of viral infections. In the case of SARS-CoV-2, combinations of multiple therapeutic antibodies may need to be rapidly identified and formulated in a way that blocks each new, predominant variant of the virus. For efficient introduction of any new antibody combination into patients, it is important to be able to monitor patient-specific pharmacokinetics of individual antibodies, which would include the time course of their specific capacity to block the viral spike proteins. Here, we present three examples of microfluidic-based rapid isolation of companion reagents useful for establishing combination antibody therapies. These reagents are specific three-dimensional imprints of variable regions of individual human monoclonal antibodies against the -spike protein of SARS-CoV-2 virus in the form of oligonucleotide-based ligands (aptamers). We implement these anti-idiotypic aptamers as bioreceptors in graphene-based field-effect transistor sensors to accomplish label free, rapid, and sensitive detection of matching antibodies within minutes. Through this work we have demonstrated the general applicability of anti-idiotype aptamers as capture reagents in quantification of active forms of monoclonal antibodies in complex biological mixtures.

Development of a fusion partner cell line for efficient production of human monoclonal antibodies from peripheral blood lymphocytes.

We developed a unique fusion partner cell line that is capable of fusing with both human peripheral blood and lymph node lymphocytes at a high efficiency. The cell line was generated by fusing a murine myeloma cell line with a human myeloma cell line, producing a heteromyeloma (B6B11), which was subsequently fused with a human lymph node lymphocyte to produce a trioma (MFP-2). B6B11 and MFP-2 fuse well with human lymphocytes from both spleen and lymph nodes. Interestingly, MFP-2 also fuses with a high efficiency to peripheral blood lymphocytes. The resulting hybrids are stable for extended periods of time and produce human monoclonal antibodies at significant levels. The utility of MFP-2 as a fusion partner was demonstrated by the isolation of several hybridoma cell lines using lymph node and peripheral blood lymphocytes from patients with breast cancer. These hybridomas produce monoclonal antibodies displaying specificity to breast cancer tissue and cell lines.

Isolation of native human monoclonal autoantibodies to breast cancer.

Using a unique fusion partner cell line, MFP-2, and B-lymphocytes from breast cancer patients, we developed a set of fully human monoclonal antibodies (MAbs) that bind with high specificity and sensitivity to breast cancer cells. Immunofluorescent staining of normal tissues, primary tumors, and metastatic lymph nodes demonstrates that these antibodies are specific for breast cancer of autologous and allogeneic origin. We have also determined that many of the antibodies selected based on specific binding to breast cancer cells and tissue also bind prostate cancer cells and tissue with high specificity and sensitivity. The targets of these antibodies have been localized to the cytoplasm and membrane. Biological assays for internalization and cytotoxicity demonstrated the ability of three antibodies to rapidly internalize. Our study demonstrates that isolation of native human MAbs from the natural antibody repertoire, targeted to cancer cells, is feasible and may provide a source of tools for immunotherapy.