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Lymphomas account for about 4% of all cancer diagnosis in the United States annually. The most common form of Non-Hodgkin’s lymphoma is known as diffuse large B-cell lymphoma (DLBCL). A frequently used treatment regimen against these lymphatic cancers involve a chemotherapy combination of cyclophosphamide, vincristine, doxorubicin, and prednisone, collectively known as CHOP. In addition to CHOP, the monoclonal antibody Rituximab has been shown to be an effective treatment against DLBCL, targeting the pan B-cell marker CD20. These therapies combined, known as R-CHOP, have been highly effective at treating DLBCL. However, 30-40% of DLBCL patients experience either relapse after R-CHOP treatment or have refractory disease. Mechanisms behind Rituximab resistance are not fully understood. Presumably, one cause for resistance is due to a decrease or loss of CD20 expression by lymphoma cells, in turn causing Rituximab to be less effective against these new cell populations.

We used single cell genomic analysis of B cells to identify alternative cell surface receptors that could be targets for broader immunotherapy. We identified two surface receptors, CD74 and IL-4 Receptor (IL-4R), that were more broadly expressed on all B cell populations as well as CD20 negative B cell populations. CD74 and IL4R were also found to be highly expressed in lymphoma tumor samples by gene expression analysis.

Two monoclonal antibodies are currently in clinical study against CD74 and IL4R, Milatuzumab and Dupilumab respectively. We developed bispecific antibodies to target both CD20 and CD74 together, and CD20 and IL-4R together. To determine the effectiveness of the antibodies, 3 lymphoma cell lines were used: Rituximab-sensitive SU-DHL-4, an intermediate Rituximab-resistant NU-DHL-1, and Rituximab-resistant SU-DHL-8. The individual CD74 and IL-4R antibodies showed no Antibody-dependent cellular cytotoxicity (ADCC) or Antibody-dependent cellular phagocytosis (ADCP), however the bispecific antibodies did have responses to the cell lines for both ADCC and ADCP. The ability of the antibodies to produce apoptosis was measured through Annexin V staining with an additional crosslinking antibody. The bispecific CD20/CD74 antibody and Rituximab had comparable apoptosis to Rituximab-sensitive SU-DHL-4 cells. Interestingly, the bispecific CD20/CD74 antibody exhibited increased apoptosis among Rituximab-resistant SU-DHL-8 cells compared to treatment with Rituximab. This finding provides evidence towards a possible additional therapeutic target of CD74 to help combat treatment resistance among lymphoma patients. These experimental discoveries open the door for the potential of using single cell genetic analysis to assist in identifying future therapeutic targets for not only lymphoma and cancer, but other diseases as well.

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Development Of Bispecific Antibodies Against Rituximab Resistant Large B Cell Lymphoma



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