title: Specific targeting of lymphoma cells using semisynthetic anti-idiotype shark antibodies
Creators: name: Macarrón Palacios, Arturo
Publisher: name: UNSPECIFIED
Year: 2021

description: The present thesis describes the versatile generation and characterization of anti-idiotype shark-derived vNAR antibodies for the development of personalized lymphoma treatment strategies. This approach relies on the B cell receptor (BCR) as a functionally active and tumor-specific cell surface marker. Since its variable region, referred to as the idiotype (Id), is unique for each B cell clonal population, this molecule differs from BCRs expressed by non-cancerous B lymphocytes but also from BCRs expressed by tumor cells of other patients. Current standard front-line treatment of several non-Hodgkin lymphomas and other hematological malignancies includes a combination of extensive chemotherapy with CD20-targeting monoclonal antibodies. Despite its high efficacy, these therapies involve multiple disadvantages derived from the concomitant depletion of all B lymphocytes of a patient and the resulting profound suppression of the whole immune system. Notwithstanding high initial response rates, most patients eventually relapse and succumb to their disease as a consequence of incomplete depletion of cancerous cells and subsequent treatment regimen resistance. Ever since the first attempts of using anti-idiotype antibodies as targeted therapy for hematological neoplasms over 30 years ago, dramatic technological advances have been made. Promising results have been achieved upon anti-idiotype antibody therapy in vivo, leading even to complete regression of lymphoma patients in clinical studies. Yet, the consequent need of developing a custom-tailored therapy represents a serious technical challenge that impeded the clinical utility of monoclonal anti-idiotypic antibodies as standard of care. Due to the singular attributes of heavy-chain only antibodies from the cartilaginous fish, harnessing these molecules has emerged as an auspicious strategy for the generation of novel therapeutics. During previous work conducted in our research group, various vNAR domains specifically targeting the IgM λ B cell receptor of the human Burkitt lymphoma cell line SUP-B8 were isolated via FACS-based screening of a semi-synthetic, CDR3-randomized yeast surface display vNAR library. After expression of putative lead candidates as vNAR-Fc fusion proteins and its characterization, various strategies were evaluated within the scope of the present thesis for specific killing of malignant B cells. First, we aimed at the stimulation of lymphoma cell apoptosis upon cell surface BCR clustering by means of anti-idiotype vNAR-Fc fusion antibodies. Several antibody formats comprising varying valences, spatial configuration, interdomain spacing, and Fc-related characteristics were designed revealing, however, no antiproliferative effects on cancerous cells. Consequently, we next sought the specific redirection of immune effector cells towards cancer cells. Anti-idiotype bispecific antibodies targeting either FcRI (CD89) expressed by neutrophils and other effector cell populations, or the T cell-related CD3 receptor revealed suitable binding properties and robust T cell activation potential but were finally unable to mediate anti-tumor activity. Monospecific, bivalent vNAR-Fc antibodies also failed at inducing cellular cytotoxicity or phagocytosis via Fc- dependent recruitment of NK cells and macrophages, respectively. As an alternative, we intended the generation of shark-derived antibody-drug conjugates (ADCs). Enzymatic MMAE-conjugation of two vNAR-Fc lead candidates resulted in reproducibly specific lysis of Burkitt cells at picomolar concentrations. Due to the outstanding specificity mediated by the vNAR domains, ADC treatment showed no cytotoxic effects on unrelated CHO cells and on off-target lymphoma cell lines in the same dose range. Hence, this approach offers a wide therapeutic window for personalized lymphoma treatment while preventing overall immunosuppression derived from the ablation of the whole B cell subset in patients. This work demonstrates the feasibility of generating shark-derived immunotherapeutics against lymphoma cells unique to each patient in a cost and time effective manner. Future investigations will evaluate the feasibility of harnessing shark-derived antibodies for the development of further toxin-free personalized therapies.